Anti-cancer compounds targeting ral gtpases and methods of using the same

ABSTRACT

Methods of inhibiting the growth or metastasis of a cancer in a subject by inhibiting a Ral GTPase in the subject, and small molecule inhibitors of Ral GTPases useful in the methods of the invention. Pharmaceutical compositions containing the compounds of the invention, and methods of using the same.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/324,629, filed Jan. 6, 2017, which is a national stage applicationunder 35 U.S.C. 371 of PCT Application No. PCT/US2015/040021 having aninternational filing date of Jul. 10, 2015, which designated the UnitedStates, which PCT application claimed the benefit of U.S. ApplicationSer. No. 62/022,946, filed Jul. 10, 2014, both of which are incorporatedherein by reference in their entirety.

GOVERNMENT INTEREST

This invention was made with Government support under grant numbersCA091846, CA075115 and CA104106 awarded by the National Institutes ofHealth. The U.S. Government has certain rights in the invention.

TECHNICAL FIELD

The invention relates to therapeutic compounds, pharmaceuticalcompositions containing the same and their use in the treatment ofcancer.

BACKGROUND OF INVENTION

Ras is mutated in cancer more frequently than any other oncogene. Hence,Ras has been a focus for the development of rationally designedanti-cancer drugs, yet to date none have been successfully developed. In1989, several groups showed that posttranslational modification of Rasproteins by farnesyl lipids is essential for Ras membrane associationand transformation. Farnesyltransferase (FTase) was then purified andcharacterized and shortly thereafter, a second prenyltransferase,geranylgeranyltransferase type I (GGTase-I), that modifies Ras with ageranylgeranyl lipid was discovered. GGTase-I inhibitors (GGTIs) werestudied and at least one such inhibitor, GGTI-2417, has been shown toinhibit the in vitro growth and survival of the MiaPaCa2 pancreatic cellline. But these inhibitory effects were modest and no clinical trialswith GGTIs have followed.

Ral (Ras-like) GTPases are members of the Ras superfamily of GTPases,and function as molecular switches that cycle between the activeGTP-bound an inactive GDP-bound states, becoming activated uponinteraction with one of a family of Ral-specific guanine nucleotideexchange factors (Ral-GEFs), which promote GDP release from Ral allowingGTP to bind in its place. Ral-GEFs, along with Raf andphosphoinositide-3-kinase (PI3-K) constitute the three known classes ofproteins whose activities are regulated by binding to Ras proteins incells. Ral-GTPases share 46% -51% identity with human Ras, are animportant component of Ras signaling and Ras oncogenesis and are animportant effector of mutant Ras in tumors (Genes & Cancer 20112(3):275-287). Ral GTPases are also highly implicated in tumormetastasis, which is the major cause of death in cancer patients. Ralproteins are therefore clinically important targets for therapeuticintervention similar to Ras. But failure to obtain clinically usefulinhibitors for Ras or any other GTPases suggests this target family is atherapeutic challenge. One reason for this is the inability to directlytarget the active site of small G proteins for inhibition because oftheir high affinity for the guanine nucleotides GDP/GTP and themillimolar concentration of these nucleotides in cells. Unlike Ras andother GTPases, however, RalA or RalB mutations are rare (<1%) in humancancer or cancer cell lines making the targeting of Ral a viableapproach to developing effective anti-cancer therapeutics.

Thus, Ral GTPases present a compelling therapeutic target for theprevention and treatment of solid tumors and the metastasis of thesecancers, and there exists a need for effective methods of inhibiting RalGTPases for the treatment of cancer.

SUMMARY OF INVENTION

The present invention provides small molecules that bind to andeffectively inhibit Ral GTPases, and therapeutic methods of using thesame. The inventors' discovery was based on computational analysis thatidentified a site available in the inactive, but not the active, proteinconformation that is distinct from the nucleotide binding pocket.Molecular docking of small molecules to this pocket followed byexperimental verification yielded at least three compounds, whichinhibited in vitro Ral binding to its effector RalBP1, Ral mediated cellspreading in murine fibroblasts and anchorage-independent growth ofhuman cancer cell lines. Delivery of two chemically related compoundshave shown favorable pharmacokinetics and tumor drug uptake in vivo.Synthesis of derivatives of these compounds led to compounds of theinvention whose binding to RalB was confirmed by surface plasmaresonance and ¹⁵N-HSQC NMR. These compounds inhibit xenograft growth toa similar extent as siRNA Ral depletion.

The compounds of the invention inhibit the activity of both RalA andRalB equally in human tumor xenografts. Although a distinct andsometimes even antagonistic role of RalA and RalB in tumorigenesis andmetastasis has been proposed, genetic mouse models have revealedsubstantial redundancy in both development and tumorigenesis. Thesestudies support the importance and clinical utility of compounds thatinhibit both RalA and RalB GTPases.

The compounds of the invention are selective against Ral with little offtarget effects, mimicking the growth inhibition effects induced by RalsiRNA and inhibiting the activity of RalA and RalB but not the closelyrelated GTPase Ras or RhoA in xenograft tumor samples. NMR titrationexperiments showed that these compounds only bind to RalB-GDP but notRalB-GTP, thereby preventing activation by Ral-specific guaninenucleotide exchange factors (Ral-GEFs) and GDP release from Ral, withGTP binding in its place, and inhibiting Ral activity-dependentphenotypes.

This computation-based screening, followed by biochemical, cellular andin vivo assays identified the small molecules of the present inventionthat bind to and effectively inhibit the activity of Ral proteins, forclinical use in cancer therapy. Thus, the present invention providesmolecules that can inhibit Ral GTPases, as well as therapeutic uses ofthese molecules to prevent or slow the growth and metastasis of cancerin a subject. The invention also provides pharmaceutical compositionscontaining these compounds and methods of using these compounds andpharmaceutical compositions to treat or prevent cancer.

One aspect of the invention is a compound of the invention having RalGTPase inhibitory activity and having the following chemical structure:

and pharmaceutically acceptable enantiomers, diastereomers, racemates,and salts thereof, wherein:

and pharmaceutically acceptable enantiomers, diastereomers, racemates,and salts thereof, wherein:

R₁ is selected from hydrogen, halogen, —OH, —O—R₈, C₁-C₁₂ alkyl, C₃-C₁₂alkenyl, C₄-C₁₂ dienyl, C₆-C₁₂ trienyl, C₈-C₁₂ tetraenyl, C₆-C₁₂ aryl,substituted C₆-C₁₂ aryl, C₁-C₁₂-alkoxy, carboxy, cyano, C₁-C₁₂alkanoyloxy, C₁-C₁₂ alkylthio, C₁-C₁₂ alkylsulfonyl, C₂-C₁₂alkoxycarbonyl, C₂-C₁₂ alkanoylamino, S—R₈, —SO₂—R₈, —NHSO₂R₈ and—NHCO₂R₈;

R₂ is selected from hydrogen, halogen, —OH, —O—R₈, C₁-C₁₂ alkyl, C₃-C₁₂alkenyl, C₄-C₁₂ dienyl, C₆-C₁₂ trienyl, C₈-C₁₂ tetraenyl, C₆-C₁₂ aryl,substituted C₆-C₁₂ aryl, C₁-C₁₂-alkoxy, carboxy, cyano, C₁-C₁₂alkanoyloxy, C₁-C₁₂ alkylthio, C₁-C₁₂ alkylsulfonyl, C₂-C₁₂alkoxycarbonyl, C₂-C₁₂ alkanoylamino, S—R₈, —SO₂—R₈, —NHSO₂R₈ and—NHCO₂R₈;

R₃, R₄, R₅, R₆, and R₇ are independently selected from hydrogen,halogen, —OH, —O—R₈, C₁-C₁₂ alkyl, C₃-C₁₂ alkenyl, C₄-C₁₂ dienyl, C₆-C₁₂trienyl, C₈-C₁₂ tetraenyl, imidazole, C₆-C₁₂ aryl, C₁-C₁₂-alkoxy,carboxy, cyano, C₁-C₁₂ alkanoyloxy, C₁-C₁₂ alkylthio, C₁-C₁₂alkylsulfonyl, C₂-C₁₂ alkoxycarbonyl, C₂-C₁₂ alkanoylamino, S—R₈,—SO₂—R₈, —NHSO₂R₈, —NHCO₂R₈, C₁-C₁₂ alkyl optionally substituted withone to three groups selected from halogen, oxygen C₁-C₆ alkyl, C₆-C₁₀aryl, and C₁-C₆ alkoxy, and C₆-C₁₂ aryl optionally substituted with oneto three groups selected from halogen, C₁-C₆ alkyl, C₆-C₁₀ aryl, andC₁-C₆ alkoxy; or,

R₃ and R₄ together form cyclohexane, 1,4-dioxane, or phenyl; and,

R₈ is C₁-C₁₂ alkyl optionally substituted with one to three groupsselected from halogen, oxygen, C₁-C₆ alkyl, C₆-C₁₀ aryl, and C₁-C₆alkoxy, or C₆-C₁₂ aryl optionally substituted with one to three groupsselected from halogen, C₁-C₆ alkyl, C₆-C₁₀ aryl, and C₁-C₆ alkoxy.

In certain embodiments, the R₁ substituent of the compound is selectedfrom hydrogen, methyl, phenyl, methyl-phenyl, methoxy, C₆-C₁₂ arylsubstituted with one to three groups selected from halogen, C₁-C₆ alkyl,and C₁-C₆ alkoxy.

In certain embodiments, the R₂ substituent of the compound is selectedfrom hydrogen, methyl, phenyl, methyl-phenyl, methoxy, C₆-C₁₂ arylsubstituted with one to three groups selected from halogen, C₁-C₆ alkyl,and C₁-C₆ alkoxy.

In certain embodiments, the R₃ substituent of the compound is selectedfrom hydrogen, halogen, methoxy, C₁-C₆ alkyl optionally substituted withhalogen, cyano, imidazole, and C₆-C₁₂ aryl substituted with one to threegroups selected from halogen, and C₁-C₆ alkoxy.

In specific embodiments, the compound has a chemical structure selectedfrom:

6-amino-1, 3-dimethyl-4-(4-(trifluoromethyl)phenyl)-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile,

6-amino-1-methyl-3-phenyl-4-(4-(trifluoromethoxy)phenyl)-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile,

6-amino-4-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3-dimethyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile, and,

6-amino-4-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-methyl-3-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile.

Another aspect of the invention is a method of treating a cancer byadministering to a subject in need of such treatment, atherapeutically-effective amount of a compound that inhibits Ral GTPaseenzymatic activity. In one aspect of this embodiment, the compoundinhibits at least one paralog of Ral GTPAse (either RalA or RalB),thereby inhibiting the growth or metastasis of a cancer. In a preferredaspect of this embodiment, the compound inhibits both RalA and RalBparalogs.

In a specific embodiment of these methods of treating or preventing acancer in a subject, the compound is administered to the subject withina pharmaceutical composition of the invention.

Thus, another aspect of the invention is a pharmaceutical compositioncontaining one or more of the compounds of the invention with at leastone pharmaceutically-acceptable carrier.

Another embodiment of the invention is a method of preventing ortreating metastatic cancers, particularly metastatic pancreas, prostate,lung, bladder, skin and/or colon cancers, by administering atherapeutically effective amount of at least one compound of theinvention to a subject in need of such treatment or suspected of havinga cancer or a metastasis of a cancer.

Another embodiment of the invention is a method of treating cancer byadministering a therapeutically effective combination of at least one ofthe compounds of the invention and one or more other known anti-canceror anti-inflammatory treatments. For example, other anti-cancertreatments may include prenyltransferase inhibitors, includinggeranylgeranyltransferase type I (GGTase-I) inhibitors, surgery,chemotherapy, radiation, immunotherapy, or combinations thereof.

Also provided herein are methods for the prevention, treatment orprophylaxis of cancer in a subject comprising administering to a subjectin need thereof, therapeutically-effective amounts of any of thepharmaceutical compositions of the invention.

Also provided herein are methods for preventing the metastasis of acancer in a subject comprising administering to the subject,therapeutically-effective amounts of at least one compound of theinvention, including, for example, pharmaceutical compositionscontaining at least one compound of the invention.

Also provided herein are pharmaceutical packages comprisingtherapeutically-effective amounts of at least one compound of theinvention within a pharmaceutical composition. The pharmaceuticalcompositions may be administered separately, simultaneously orsequentially with other compounds or therapies used in the prevention,treatment or amelioration of cancer in a subject. These packages mayalso include prescribing information and/or a container. If present, theprescribing information may describe the administration, and/or use ofthese pharmaceutical compositions alone or in combination with othertherapies used in the prevention, treatment or amelioration of cancer ina subject.

Another embodiment of this invention is a method of testing thesusceptibility of a subject having lung cancer to treatment with aputative inhibitor of Ral GTPase activity by testing the subject for aresponse to administration of the putative inhibitor indicative ofgrowth inhibition or reduction in cancer cell number or tumor volume inthe subject.

Other aspects of the invention will be set forth in the accompanyingdescription of embodiments, which follows and will be apparent from thedescription or may be learned by the practice of the invention. However,it should be understood that the following description of embodiments isgiven by way of illustration only since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art and are encompassed within thescope of this invention.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1D show the molecular modeling of the target site on Ralprotein. Structural model of RalA-GDP in ribbon (FIG. 1A) or surface(FIG. 1B) representations. The allosteric binding site is formed byswitch II, helix α2 and helix α3. (C-D) Surface representations ofRalA-GNP in complex with exo84 (FIG. 1C, exo84 not shown), and RalA-GNPin complex with sec5 (FIG. 1D, sec5 not shown). In FIGS. 1B and 1C, thesphere/surfaces indicate the water-accessible area in the bindingcavity. All models were generated in Accelrys Discovery Studio softwareusing published structures.

FIG. 2A shows the chemical synthesis schemes for compounds BQU57 andBQU85. Figure S3B-D shows the characterization of BQU57 binding to Ral.FIG. 2B shows the chemical shift changes in RalB-GNP (100 μM) in thepresence of 100 μM BQU57. FIG. 2C shows the plot of 1H-15N-HSQC NMRchemical shift changes of selected residues in RalB-GDP with increasingconcentrations of BQU57. Surface plasmon resonance determination of KDfor binding between BQU57 and RalB-GDP showed a fitted binding curvegiving a KD value of 4.7 μM.

FIGS. 3A-3E show the characterization of compounds binding to Ral. FIG.3A shows the structure of BQU57, a derivative of RBCS. FIG. 3B is anoverlay of the ¹⁵N-HSQC spectrum of 100 μM Ral-GDP and in the presenceof 100 μM BQU57. FIG. 3C shows selected residues of RalB-GDP in theabsence and presence of increasing concentrations of BQU57 at 40 μM and100 μM. FIG. 3D shows a plot of chemical shift changes as a function ofresidue number comparing RalB-GDP alone (100 μM) and in the presence of100 μM BQU57. FIG. 3E shows the binding of BQU57 to RalB-GDP determinedusing Isothermal Titration calorimetry (ITC).

FIGS. 4A-4J shows the growth inhibitory activity of Ral inhibitors inhuman cancer cell lines. Effect of BUQ57 (FIG. 4A) and BQU85 (FIG. 4B)treatment on the anchorage-independent growth of four human lung cancercell lines. Cells were seeded in soft agar containing variousconcentrations of drug; colonies formed in soft agar were counted after2-4 weeks. Cell lines that are sensitive to Ral siRNA knockdown (H2122and H358) are colored gray and cell lines resistant to Ral siRNAknockdown (H460 and Calu6) are colored black. Data represents the meanof three independent experiments. Effect of siRNA knockdown of both RalAand RalB in H2122 (FIGS. 4C, 4D) and H358 (FIGS. 4E, 4F) cells ondrug-induced growth inhibition in soft agar. Cells were transfected with10/30/50 nM of siRNA for 48 h, collected, and subjected to the soft-agarcolony formation assay. Effect of siRNA alone on soft agar colony numberis shown in FIG. 4C (H2122) and FIG. 4E (H358); effect of siRNA plusdrug treatment on colony formation is shown as percent of DMSO treatedcontrol in FIG. 4D (H2122) and FIG. 4F (H358). Effect of theoverexpression of constitutively active RalA^(G23V) and RalB^(G23V) inH2122 (FIGS. 4G, 4H) and H358 (FIGS. 4I, 4J) cells on drug-inducedgrowth inhibition in soft agar. H2122 cells were transiently transfectedwith FLAG-RalA^(G23V) or FLAG-RalB^(G23V) for 48 h before the soft agarcolony formation assay. H358 cells were stably transfected withFLAG-RalA^(G23V) or FLAG-RalB^(G23V). Overexpression was confirmed byimmunoblotting and shown in FIG. 7F. All results shown represent themean±SD of three independent experiments. * denotes statisticalsignificant difference between indicated groups.

FIGS. 5A-5H show the effect of Ral inhibitors on human xenograft modelsof lung cancer. Tissue distribution of RBC8 (FIG. 5A) and BQU57 (FIG.5B) in nude mice 3 h after a single i.p. dose of 50 mg/Kg; datarepresent the mean±SD of 3 mice. (FIG. 5C and FIG. 5D) 50 mg/kg/day RBC8initiated 24 h after inoculation inhibited xenograft tumor growth of thehuman lung cancer cell line H2122. Data represents the mean±SEM of 6mice. Tumor volume in the treatment group was statistically differentfrom controls, as determined by the Dunnett's test (*p<0.05). Typicaltumor appearance shown in FIG. 5D. FIG. 5E shows siRNA depletion of bothRalA and RalB inhibited the xenograft tumor growth of H2122 cells. Cellswere transiently transfected with siRNAs against both RalA and RalB for24 h; cells were then inoculated into nude mice; tumors were monitoredand measured as described above. Data represents the mean±SEM of 6 mice.Tumor volume in the treatment group was statistically different fromcontrols as determined by the Dunnett's test (*p<0.05). FIG. 5F showsBQU57 treatment (10/20/50 mg/kg/day) initiated 24 h after inoculationinhibited xenograft tumor growth of H2122 cells. Data represents themean±SEM of 6 mice. Tumor volume in the treatment group wasstatistically different from controls as determined by the Dunnett'stest (*p<0.05). FIG. 5G shows the tissue distribution of BQU85 in nudemice 3 h after a single i.p. dose of 50 mg/Kg. Data represent themean±SD of 3 mice. FIG. 5H shows the effect of BQU85 treatment on humanxenograft models of lung cancer. BQU85 treatment (5/10/20/50 mg/kg/day)initiated 24 h after inoculation inhibited xenograft tumor growth ofH2122 cells. Data represents the mean±SEM of 6 mice.

FIGS. 6A-6E show the cellular uptake of Ral inhibitors in vitro. H2122human lung cancer cells were treated with 10 μM of RBC8 (FIGS. 6A),BQU57 (FIGS. 6B), BQU85 (FIGS. 6C), and RBCS (FIGS. 6D). Cells werecollected at different time points (1, 5, 15, 30 and 60 min), and drugconcentrations in cells determined using LC/MS-MS methods (n=3 for eachtime point). FIG. 6E shows the inhibition of Ral activity in H2122 andH358 cells by RBC5, RBC8 and BQU57. Cells were grown underanchorage-independent conditions and treated with 10₁1.M compounds for 3hours. Ral activity in cell lysates were then determined using the pulldown assay with RalBP1 agarose beads. Data represent three independentexperiments.

FIGS. 7A-7F show the effect of K-Ras or Ral knockdown or overexpressionon anchorage-independent growth of four human lung cancer cell lines.FIG. 7A shows an immunoblot of siRNA knockdown of K-Ras in H2122, H358,H460, and Calu6 cell lines 48 h after siRNA transfection. FIG. 7B showsthat all four lines were sensitive to K-Ras knockdown using the softagar colony formation assay. The effect of Ral knockdown onanchorage-independent growth of four human lung cancer cell lines wasinvestigated by transfecting the cells with siRNA against RalA, RalB orRalA/B for 48 h and then subjecting the cells to soft agar colonyformation assays. FIG. 7C shows that cell lines H2122/H358 weresensitive to Ral knockdown. FIG. 7D shows that cell lines H460/Calu6were not sensitive to Ral knockdown. FIG. 7E shows immunoblots ofknockdown of both RalA and RalB in H2122 and H358 cell lines 48 h aftertreatment with various concentrations of siRNA. FIG. 7F showsimmunoblots of successful overexpression of constitutively activeRalA^(G23V) and RalB^(G23V) in H2122 and H358 cells. H2122 cells weretransiently transfected with FLAG, FLAG-RalA^(G23V) and FLAG-RalB^(G23V)for 48h. H358 cells stably overexpressing FLAG, FLAG-RalA^(G23V) andFLAG-RalB^(G23V) were generated by G418 selection.

DESCRIPTION OF EMBODIMENTS

Based on compelling clinical significance in tumor establishment andmetastasis, the present inventors have identified and used Ral GTPasesas molecular targets. As with all GTPases, activity of Ral is dependentupon cycling between an inactive (GDP-bound) and an active (GTP-bound)conformation. Active Ral proteins mediate downstream processes throughtheir own set of effectors, including Ral Binding Protein 1 (RalBP1,RLIP76 or RIP1(37)), Sec5/Exo85, filamin, and phospholipase D1. Thus,compounds that bind Ral-GDP and not Ral-GTP may be used to stericallyinhibit effector binding and/or block conformational changes associatedwith the GTP bound state, leading to blockade of signal transmissionwith consequent decreased growth and apoptosis of Ral-dependent cancercells. These compounds were identified using both virtual and physicalscreening of Ral GTPase inhibitors.

As noted above, Ral cycles between inactive (GDP-bound) and active(GTP-bound) forms. With the goal of finding compounds thatpreferentially bind to Ral-GDP (inactive) over Ral-GTP (active) andthereby stabilize Ral in the inactive state, the inventors inspected thethree-dimensional structures of RalA in its active and inactive forms.This analysis revealed differences in the shape of a pocket near, butdistinct from the nucleotide binding site (FIG. 1). This pocket(allosteric site) is similar to the previously described C3bot bindingsite and is made up by the switch-II region (Ral70-Ral77), helix α2(Ral78-Ral85) and one face of helix α3 (FIG. 1A). The crystal structuresused in the comparison included RalA-GDP (PDB code 2BOV (FIG. 1B) andRalA-GNP (non-hydrolysable form of GTP) in complex with exo84 (PDB code1ZC4, FIG. 1C) or sec5 (PDB code 1UAD, FIG. 1D). Volumes calculated foreach binding site were 175 Å³ for RalA-GDP (FIG. 1B), 155 Å³ forRalA-GNP-exo84 (FIG. 1C), and 116 Å³ for RalA-GNP-sec5 (FIG. 1D). TheRalB-GDP crystal structure is not published, but in the RalB-GNPstructure (PDB code 2KE5, FIG. 1) this binding pocket is almost absent.Using a structure-based virtual screening approach to identify smallmolecules that bind to the allosteric site of RalA-GDP, 500,000compounds were docked to the RalA-GDP pocket. The protein-ligandcomplexes were scored and sorted based on the calculated interactionenergies followed by visual inspection of top candidates which led tothe selection of 88 compounds. The 88 selected compounds were evaluatedfor their ability to inhibit RalA activation in living cells in cultureusing an ELISA for Ral activity based on selective binding of activeRalA-GTP to its effector protein RalBP1.

RalA activity was also assayed independently by measuring lipid raftexocytosis during spreading of murine embryonic fibroblasts (MEFs) onfibronectin-coated coverslips. In these cells, siRNA depletion of RalAinhibits spreading, whereas caveolin (Cav1)-/- MEFs are resistant toRalA depletion.

TROSY¹⁵N-HSQC (Transverse Relaxation-Optimized Heteronuclear SingleQuantum Coherence) NMR was used to confirm the direct binding of thecompounds to the Ral target site. The inventors focused on NMR structureof RalB in complex with GNP (the only structure that has been solved atthis time). The ¹⁵N-HSQC NMR spectrum of RalB-GDP and RalB-GNP werefirst determined and the chemical shift difference was analyzed. NMRspectra were then recorded in the presence of RBC8 or DMSO control.Binding of small molecules to the protein was monitored by theperturbation of ¹⁵N-HSQC protein amide peaks. The 15N-HSQC spectrum ofRalB-GDP (100 μM) in the absence and presence of 100 μM RBC8, showedchanges in peak position of representative residues located in theallosteric site. RBC8 did not bind to RalB-GNP under the same conditionsas indicated by minimal chemical shift changes on the NMR spectrum.Moreover, RBC5, which did not affect the level of active Ral in thecell-based ELISA assay, also did not induce chemical shift changes inRalB-GDP, therefore serving as additional negative control.

Based on all data including structural features, a series of RBC8derivatives was synthesized and tested for binding in vitro. BQU57 waschosen for further evaluation because of its superior performancecompared to RBC8 and its drug-like properties. A detailed NMR analysisof the binding between BQU57 and RalB-GDP was carried out. A plot of thechemical shift changes with BQU57 as a function of sequence showed thatresidues that exhibit significant changes are located in the switch-II(amino acid residues 70-77) and helix α2 (amino acid residues 78-85)region. Because no RalB-GDP crystal structure is available, a homologymodel was generated based on the similarity to RalA-GDP, and theresidues that displayed chemical shift changes in response to thecompounds were mapped onto this model. The majority of the chemicalshift changes localized to the allosteric site, consistent withassignment of BQU57 binding to this site based on modeling. Similar toresults with RBC8, BQU57 did not bind to RalB-GNP (100 μM) as indicatedby minimal chemical shift changes on NMR spectrum. Analysis of the NMRchemical shift titrations revealed that binding of BQU57 wasstoichiometric up to the apparent limiting solubility of the drug. Thebinding of BQU57 to RalB-GDP was also determined using IsothermalTitration calorimetry (ITC) and the results were similar to results fromSurface Plasma Resonance (SPR).

The effects of RBC8 and BQU57 on human lung cancer cell growth wereevaluated. Because Ral is well-known for its role in anchorageindependence the inventors carried out growth inhibition assays in softagar. Human lung cancer cells were used in a series of experiments todetermine drug uptake, biologic specificity, and effect.

The cellular uptake of RBC8, BQU57, BQU85, and RBCS was examined and allcompounds were found to readily get into cells. All cell lines werefound to be sensitive to K-Ras siRNA depletion but only H2122 and H358were sensitive to Ral knockdown. Using this characteristic to determinethe specificity of the compounds to Ral compared to Ras, a closelyrelated GTPase, the inventors evaluated inhibition of colony formationin soft agar and found the Ral-dependent lines H2122 and H358 weresensitive. Additionally, a Ral pull-down assay using RalBP I agarosebeads showed that RBC8 and BQU57, but not RBC5, inhibited both RalA andRalB activation in both the H2122 and H358 cell lines. A chemo-genomicexperiment was performed to further determine drug specificity to Ral.Treatment of H2122 and H358 cells that had siRNA knockdown of RalA andRalB with RBC8 or BQU57 did not result in significant furtherinhibition. Together, these data demonstrated RBC8 and BQU57 reduceanchorage independent growth via Ral inhibition.

The specificity of the compounds for the GDP form, compared to the GTPform of Ral, was evaluated by constitutively overexpressing the activeform of RalAG23V or RalBG23V in H2122 and H358 cells. (The G23V mutationprevents RalGAP mediated activation of GTP hydrolysis and hence locksRal in its active state.) Both RalAG23V and RalBG23V could rescue thegrowth inhibition effect of RBC8 and BQU57 compounds.

Inhibition of Ral activity and tumor growth were evaluated in human lungcancer mouse models. Pharmacokinetics of RBC8 and BQU57 were firstanalyzed in mice to test bioavailability, with RBC8 and BQU57 showingfavorable properties that define good drug candidates, as shown in Table1.

TABLE 1 Pharmacokinetic characteristics of selected compounds. RBC8BQU57 Dose (mg/kg) i.p. n = 3 50.0 50.0 Co (μM) 41.2 ± 4.2  41.6 ± 5.1T_(1/2) (hr) 0.58 ± 0.26  1.50 ± 0.11 AUC_(0-5 hr) (mg · h/mL) 139.6 ±18.8  28.6 ± 2.1

Compound entry into tumor tissue was determined and substantial amountsof compound were detected in tumor tissue 3 hours post-dose.

The effect of the Ral inhibitors on xenograft tumor growth was thentested in nude mice. RBC8 inhibited tumor growth by the same order ofmagnitude as dual knockdown of RalA and RalB, and a second lung cancerline, H358 yielded similar results. BQU57 and BQU85 were also tested invivo and dose-dependent growth inhibition effects were observed.

Ral GTPase activity was evaluated in vivo in the H2122 xenografts.RalBP1 pull-down measurements of Ral activity showed significantinhibition of both RalA and RalB by RBC8 and BQU57. Importantly,BQU57-induced dose-dependent inhibition of Ral activity correlated withinhibition of tumor growth. Additionally, Ras and RhoA activity wasmeasured in BQU57 treated tumors and no significant inhibition wasobserved, further demonstrating the selectivity of the Ral inhibitors ofthe invention.

Hence, the present invention provides Ral GTPase inhibiting compounds.These compounds can bind to the inactive form of Ral protein and preventGEF-induced activation or GTP exchange and are selective against Ralwith little off target effects. Thus, the Ral GTPase inhibitors of thisdisclosure can be used to block the associated conformational change ofRal proteins upon GTP binding, thus preventing effector engagement anddownstream signaling.

Thus, the present invention also provides methods of inhibiting thegrowth and/or metastasis of cancer in a subject by inhibiting a RalGTPase in the subject. In a preferred embodiment, the Ral GTPase is atleast one of the RalA and the RalB paralogs. The term “paralog” is usedin this disclosure to denote genes in an organism that have beenduplicated to occupy different positions in the same genome.

In another aspect, the invention provides a method of inhibiting thegrowth and/or metastasis of cancer in a subject by administering atleast one compound of the invention, or pharmaceutically-acceptablesalts thereof to the subject.

As used herein, the term “compound” means a chemical or biologicalmolecule such as a simple or complex organic molecule, a peptide, aprotein or an oligonucleotide.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complicationcommensurate with a reasonable benefit/risk ratio.

“Pharmaceutically-acceptable salts” refer to derivatives of thedisclosed compounds wherein the parent compound is modified by makingacid or base salts thereof. Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines, or alkali or organic salts of acidicresidues such as carboxylic acids. Pharmaceutically-acceptable saltsinclude the conventional non-toxic salts or the quaternary ammoniumsalts of the parent compound formed, for example, from non-toxicinorganic or organic acids. Such conventional nontoxic salts includethose derived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfamic, phosphoric, nitric and the like; and the saltsprepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic,ethane disulfonic, oxalic, isethionic, and the like. Pharmaceuticallyacceptable salts are those forms of compounds, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problems orcomplications, commensurate with a reasonable benefit/risk ratio.

Pharmaceutically-acceptable salt forms of compounds provided herein aresynthesized from the compounds of the invention which contain a basic oracidic moiety by conventional chemical methods. Generally, such saltsare, for example, prepared by reacting the free acid or base forms ofthese compounds with a stoichiometric amount of the appropriate base oracid in water or in an organic solvent, or in a mixture of the two;generally, nonaqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are preferred. Lists of suitable salts arefound in at page 1418 of Remington's Pharmaceutical Sciences, 17th ed.,Mack Publishing Company, Easton, Pa., 1985.

The term “subject” refers to mammals such as humans or primates, such asapes, monkeys, orangutans, baboons, gibbons, and chimpanzees. The term“subject” can also refer to companion animals, e.g., dogs and cats; zooanimals; equids, e.g., horses; food animals, e.g., cows, pigs, andsheep; and disease model animals, e.g., rabbits, mice, and rats. Thesubject can be a human or non-human. The subject can be of any age. Forexample, in some embodiments, the subject is a human infant, i.e., postnatal to about 1 year old; a human child, i.e., a human between about 1year old and 12 years old; a pubertal human, i.e., a human between about12 years old and 18 years old; or an adult human, i.e., a human olderthan about 18 years old. In some embodiments, the subject is an adult,either male or female.

The term “therapeutically-effective amount” or “therapeutic amount” of acompound of this invention means an amount effective to inhibit theformation or progression of cancer following administration to a subjecthaving a cancer.

The term “solvate” refers to the compound formed by the interaction of asolvent and a compound. Suitable solvates are pharmaceuticallyacceptable solvates, such as hydrates, including monohydrates andhemi-hydrates.

It will be appreciated by those skilled in the art that compounds of theinvention having a chiral center may exist in, and may be isolated in,optically active and racemic forms. It is to be understood that thecompounds of the present invention encompass any racemic,optically-active, regioisomeric or stereoisomeric form, or mixturesthereof, which possess the therapeutically useful properties describedherein. Where the compounds of the invention have at least one chiralcenter, they may exist as enantiomers. Where the compounds possess twoor more chiral centers, they may additionally exist as diastereomers.Where the processes for the preparation of the compounds according tothe invention give rise to mixtures of stereoisomers, these isomers maybe separated by conventional techniques such as preparativechromatography. The compounds may be prepared in racemic form or asindividual enantiomers or diasteromers by either stereospecificsynthesis or by resolution. The compounds may, for example, be resolvedinto their component enantiomers or diasteromers by standard techniques,such as the formation of stereoisomeric pairs by salt formation with anoptically active acid, such as (−)-di-p-toluoyl-D-tartaric acid and/or(+)-di-p-toluoyl-L-tartaric acid followed by fractional crystallizationand regeneration of the free base. The compounds may also be resolved byformation of stereoisomeric esters or amides, followed bychromatographic separation and removal of the chiral auxiliary.Alternatively, the compounds may be resolved using a chiral HPLC column.It is to be understood that all stereoisomers, racemic mixtures,diastereomers and enantiomers thereof are encompassed within the scopeof the present invention.

It is well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase). It is also to be understood that the scope of thisinvention encompasses not only the various isomers, which may exist butalso the various mixtures of isomers, which may be formed. Theresolution of the compounds of the present invention, their startingmaterials and/or the intermediates may be carried out by knownprocedures, e.g., as described in the four volume compendium OpticalResolution Procedures for Chemical Compounds: Optical ResolutionInformation Center, Manhattan College, Riverdale, N.Y., and inEnantiomers, Racemates and Resolutions, Jean Jacques, Andre Collet andSamuel H. Wilen; John Wiley & Sons, Inc., New York, 1981, which isincorporated in its entirety by this reference. Basically, theresolution of the compounds is based on the differences in the physicalproperties of diastereomers by attachment, either chemically orenzymatically, of an enantiomerically pure moiety resulting in formsthat are separable by fractional crystallization, distillation orchromatography.

The chemicals used in combination with the compounds of the presentinvention to make the pharmaceutical compositions of the presentinvention may be purchased commercially. The compounds of the presentinvention, including the salts of these compounds, may be prepared inways well known to those skilled in the art of organic synthesis. Thecompounds of the invention may be prepared using the reactions performedin solvents appropriate to the reagents and materials employed andsuitable for the transformation being effected. It is understood by oneskilled in the art of organic synthesis that the functionality presenton various portions of the molecule must be compatible with the reagentsand reactions proposed. Such restrictions to the substituents, which arecompatible with the reaction conditions, will be readily apparent to oneskilled in the art and alternate methods must then be used.

The pharmaceutical compositions of the invention contain one or morecompounds of the invention and a pharmaceutically-acceptable carrier,which are media generally accepted in the art for the delivery ofbiologically active agents to animals, in particular, subjects.Pharmaceutically-acceptable carriers are formulated according to anumber of factors well within the purview of those of ordinary skill inthe art to determine and accommodate. These include, without limitation:the type and nature of the active agent being formulated; the subject towhich the agent-containing composition is to be administered; theintended route of administration of the composition; and, thetherapeutic indication being targeted. Pharmaceutically-acceptablecarriers include both aqueous and non-aqueous liquid media, as well as avariety of solid and semi-solid dosage forms. Such carriers can includea number of different ingredients and additives in addition to theactive agent, such additional ingredients being included in theformulation for a variety of reasons, e.g., stabilization of the activeagent, well known to those of ordinary skill in the art. Descriptions ofsuitable pharmaceutically-acceptable carriers, and factors involved intheir selection, are found in a variety of readily available sources,such as Remington's Pharmaceutical Sciences, 17th ed., Mack PublishingCompany, Easton, Pa., 1985.

This invention further provides methods of treating a subject afflictedwith a cancer or preventing the metastasis of such cancer in a subject,which includes administering to the subject a pharmaceutical compositionprovided herein. Such compositions generally comprise a therapeuticallyeffective amount of a compound of the invention in an amount effectiveto prevent, ameliorate, lessen or inhibit the cancer. Such amountstypically comprise from about 0.1 to about 100 mg of the compound perkilogram of body weight of the subject to which the composition isadministered. Therapeutically effective amounts can be administeredaccording to any dosing regimen satisfactory to those of ordinary skillin the art.

Administration may be, for example, by various parenteral means.Pharmaceutical compositions suitable for parenteral administrationinclude various aqueous media such as aqueous dextrose and salinesolutions; glycol solutions are also useful carriers, and preferablycontain a water soluble salt of the active ingredient, suitablestabilizing agents, and if necessary, buffering agents. Antioxidizingagents, such as sodium bisulfite, sodium sulfite, or ascorbic acid,either alone or in combination, are suitable stabilizing agents; alsoused are citric acid and its salts, and EDTA. In addition, parenteralsolutions can contain preservatives such as benzalkonium chloride,methyl- or propyl-paraben, and chlorobutanol.

Alternatively, compositions can be administered orally in solid dosageforms, such as capsules, tablets and powders; or in liquid forms such aselixirs, syrups, and/or suspensions. Gelatin capsules can be used tocontain the active ingredient and a suitable carrier such as, but notlimited to, lactose, starch, magnesium stearate, stearic acid, orcellulose derivatives. Similar diluents can be used to make compressedtablets. Both tablets and capsules can be manufactured as sustainedrelease products to provide for continuous release of medication over aperiod of time. Compressed tablets can be sugar-coated or film-coated tomask any unpleasant taste, or used to protect the active ingredientsfrom the atmosphere, or to allow selective disintegration of the tabletin the gastrointestinal tract.

A preferred formulation of the invention is a mono-phasic pharmaceuticalcomposition suitable for parenteral or oral administration for theprevention, treatment or prophylaxis of a cancer, consisting essentiallyof a therapeutically-effective amount of a compound of the invention,and a pharmaceutically acceptable carrier.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as wetting agents,emulsifying agents and dispersing agents. It may also be desirable toinclude isotonic agents, such as sugars, sodium chloride, and the likein the compositions. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as aluminum monosterate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which in turn may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drug isaccomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsulated matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending on the ratio of drug to polymer, and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissue. The injectable materials can be sterilized forexample, by filtration through a bacterial-retaining filter.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, 0.1 to about 500 mg of the therapeuticcompounds of the present invention.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, powders, granules or as asolution or a suspension in an aqueous or non-aqueous liquid, or anoil-in-water or water-in-oil liquid emulsions, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia), and the like, each containing a predeterminedamount of a compound or compounds of the present invention as an activeingredient. A compound or compounds of the present invention may also beadministered as bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonosterate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may be employed as fillers in soft andhard-filled gelatin capsules using such excipients as lactose or milksugars, as well as high molecular weight polyethylene glycols and thelike.

A tablet may be made by compression or molding optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions which can be used includepolymeric substances and waxes. The active ingredient can also be inmicroencapsulated form.

The tablets or pills of the present invention may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound. Formulations of thepresent invention which are suitable for vaginal administration alsoinclude pessaries, tampons, creams, gels, pastes, foams or sprayformulations containing such carriers as are known in the art to beappropriate.

Dosage forms for the topical or transdermal administration of compoundsof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches, drops and inhalants. The activeingredient may be mixed under sterile conditions with apharmaceutically-acceptable carrier, and with any buffers, orpropellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to anactive ingredient, excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to an active ingredient,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder or mixtures of these substances.Sprays can additionally contain customary propellants such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of compounds of the invention to the body. Such dosage formscan be made by dissolving, dispersing or otherwise incorporating one ormore compounds of the invention in a proper medium, such as anelastomeric matrix material. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate of such fluxcan be controlled by either providing a rate-controlling membrane ordispersing the compound in a polymer matrix or gel.

Pharmaceutical formulations include those suitable for administration byinhalation or insufflation or for nasal or intraocular administration.For administration to the upper (nasal) or lower respiratory tract byinhalation, the compounds of the invention are conveniently deliveredfrom an insufflator, nebulizer or a pressurized pack or other convenientmeans of delivering an aerosol spray. Pressurized packs may comprise asuitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, thecomposition may take the form of a dry powder, for example, a powder mixof one or more of the anti-cancer compounds of the invention and asuitable powder base, such as lactose or starch. The powder compositionmay be presented in unit dosage form in, for example, capsules orcartridges, or, e.g., gelatin or blister packs from which the powder maybe administered with the aid of an inhalator, insufflator or ametered-dose inhaler.

For intranasal administration, compounds of the invention may beadministered by means of nose drops or a liquid spray, such as by meansof a plastic bottle atomizer or metered-dose inhaler. Typical atomizersare the Mistometer (Wintrop) and Medihaler (Riker).

Drops, such as eye drops or nose drops, may be formulated with anaqueous or nonaqueous base also comprising one or more dispersingagents, solubilizing agents or suspending agents. Liquid sprays areconveniently delivered from pressurized packs. Drops can be delivered bymeans of a simple eye dropper-capped bottle or by means of a plasticbottle adapted to deliver liquid contents drop-wise by means of aspecially shaped closure.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier, for example water for injection, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the type described above.

The dosage formulations provided by this invention may contain thetherapeutic compounds of the invention, either alone or in combinationwith other therapeutically active ingredients, and pharmaceuticallyacceptable inert excipients. The term ‘pharmaceutically acceptable inertexcipients’ includes at least one of diluents, binders,lubricants/glidants, coloring agents and release modifying polymers.

Suitable antioxidants may be selected from amongst one or morepharmaceutically acceptable antioxidants known in the art. Examples ofpharmaceutically acceptable antioxidants include butylatedhydroxyanisole (BHA), sodium ascorbate, butylated hydroxytoluene (BHT),sodium sulfite, citric acid, malic acid and ascorbic acid. Theantioxidants may be present in the dosage formulations of the presentinvention at a concentration between about 0.001% to about 5%, byweight, of the dosage formulation.

Suitable chelating agents may be selected from amongst one or morechelating agents known in the art. Examples of suitable chelating agentsinclude disodium edetate (EDTA), edetic acid, citric acid andcombinations thereof. The chelating agents may be present in aconcentration between about 0.001% and about 5%, by weight, of thedosage formulation.

The dosage form may include one or more diluents such as lactose, sugar,cornstarch, modified cornstarch, mannitol, sorbitol, and/or cellulosederivatives such as wood cellulose and microcrystalline cellulose,typically in an amount within the range of from about 20% to about 80%,by weight.

The dosage form may include one or more binders in an amount of up toabout 60% w/w. Examples of suitable binders include methyl cellulose,hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, eudragits, ethyl cellulose, gelatin, gum arabic, polyvinylalcohol, pullulan, carbomer, pregelatinized starch, agar, tragacanth,sodium alginate, microcrystalline cellulose and the like.

Examples of suitable disintegrants include sodium starch glycolate,croscarmellose sodium, crospovidone, low substituted hydroxypropylcellulose, and the like. The concentration may vary from 0.1% to 15%, byweight, of the dosage form.

Examples of lubricants/glidants include colloidal silicon dioxide,stearic acid, magnesium stearate, calcium stearate, talc, hydrogenatedcastor oil, sucrose esters of fatty acid, microcrystalline wax, yellowbeeswax, white beeswax, and the like. The concentration may vary from0.1% to 15%, by weight, of the dosage form.

Release modifying polymers may be used to form extended releaseformulations containing the therapeutic compounds of the invention. Therelease modifying polymers may be either water-soluble polymers, orwater insoluble polymers. Examples of water-soluble polymers includepolyvinylpyrrolidone, hydroxy propylcellulose, hydroxypropylmethylcellulose, vinyl acetate copolymers, polyethylene oxide,polysaccharides (such as alginate, xanthan gum, etc.), methylcelluloseand mixtures thereof. Examples of water-insoluble polymers includeacrylates such as methacrylates, acrylic acid copolymers; cellulosederivatives such as ethylcellulose or cellulose acetate; polyethylene,and high molecular weight polyvinyl alcohols.

Also encompassed by the present invention are methods for screeningpotential therapeutic agents that may prevent, treat or inhibit themetastasis of lung cancer, by inhibiting a Ral GTPase comprising: (a)combining a Ral GTPase and a potential therapeutic compound underconditions in which they interact, and; (b) monitoring the enzymaticactivity of the Ral GTPase; wherein a potential therapeutic compound isselected for further study when it inhibits the enzymatic activitycompared to a control sample to which no potential therapeutic compoundhas been added. In one embodiment, the potential therapeutic compound isselected from the group consisting of a pharmaceutical agent, acytokine, a small molecule drug, a cell-permeable small molecule drug, ahormone, a combination of interleukins, a lectin, a bispecific antibody,and a peptide mimetic.

One embodiment of the invention relates to a compound of the inventionfor use in the treatment or prevention of cancer, or a metastasis of acancer, in a subject. A related embodiment of the invention relates to acomposition of the invention for use in the treatment or prevention ofcancer, or a metastasis of a cancer, in a subject.

Another embodiment of the invention relates to the use of any of thecompounds or compositions of the invention in the preparation of amedicament for the inhibition of the growth or metastasis of a cancer ina subject.

Each publication or patent cited herein is incorporated herein byreference in its entirety.

EXAMPLES

The following examples are provided to illustrate certain aspects,embodiments, and configurations of the disclosure and are not to beconstrued as limitations on the disclosure, as set forth in the appendedclaims.

Example 1—Molecular Modeling of Ral Inhibitors

Molecular modeling was used to find compounds that preferentially bindto Ral-GDP (inactive) over Ral-GTP (active) with the expectation thatsuch molecules will stabilize the inactive state. Inspection ofthree-dimensional structures of RalA in its active and inactive formsrevealed differences in the shape of a pocket near but distinct of thenucleotide binding site (FIG. 1). This pocket (allosteric site) issimilar to the previously described C3bot binding site and is made up bythe switch-II region (Ral70-Ral77), helix α2 (Ral78-Ral85) and one faceof helixα3 (FIG. 1A). The crystal structures used in the comparisonincluded RalA-GDP (PDB code 2BOV, FIG. 1B) and RalA-GNP(non-hydrolysable form of GTP) in complex with exo84 (PDB code 1ZC4,FIG. 1C) or sec5 (PDB code 1UAD, FIG. 1D). Volumes calculated for eachbinding site were 175 Å3 for RalA-GDP (FIG. 1B), 155 Å3 forRalA-GNP-exo84 (FIG. 1C), and 116 Å3 for RalA-GNP-sec5 (FIG. 1D). TheRalB-GDP crystal structure is not published, but in the RalB-GNPstructure (PDB code 2KE5, FIG. 1) this binding pocket is almost absent.

We followed a structure-based virtual screening approach to identifysmall molecules that bind to the allosteric site of RalA-GDP. Thecrystallographic coordinates of the 2.66 Å human RalA-GDP (PDB: 2BOV),RalA-GNP in complex with exo84 (PDB: 1ZC4), RalA-GNP in complex withsec5 (PDB: 1UAD) crystal structures were obtained from the RCSB ProteinData Bank (rcsb.org). AutoDock4 was used for the initial libraryscreening. The ChemDiv library [v2006.5, 500,000 compounds excludingthose possessing reactive groups, known ADME/toxicity, physicochemicalproperties lie outside ‘drug-likeness’ parameters (Lipinski's rule of 5and Veber's Rule of 2) at pH 7] was downloaded from ZINC database anddocked into the identified site on RalA-GDP using rigid dockingprotocols. Ligand molecules were assigned Gasteiger charges and polarhydrogen atoms by the ligand preparation module provided in theAutoDockTools. The Lamarckian genetic algorithm in AutoDock4 was used toevaluate ligand binding energies over the conformational search space.Protein-ligand complexes were scored and sorted based on the calculatedinteraction energies followed by visual inspection of top candidateswhich led to selection of 88 compounds.

Example 2—Cell-Based Functional Assays

The 88 selected compounds were evaluated for their ability to inhibitRalA activation in living cells in culture.

Human bladder cancer cell line J82 and lung cancer cell lines H2122,H358, H460, and Calu6 were obtained from ATCC. Antibodies used areagainst human RalA (BD Biosciences, #610222), RalB (Millipore #04-037),and FLAG tag (Novagen #71097). Activity assay kits for Ras (#BK008) andRhoA (#BK036) were obtained from Cytoskeleton (Denver, Co.). We used anELISA for Ral activity based on selective binding of active RalA-GTP toits effector protein RalBP1.

J82 cells stably overexpressing FLAG-RalA were plated 800,000 cells perwell in 6-well plates and allowed to incubate for 16 h. Cells weretreated with 500 μl of fresh medium containing test compounds (50 μM) orDMSO control (1 h; 37° C.). Cells were then washed with ice-cold PBS andcollected into ice-cold lysis buffer (750 μl containing 50 mM Tris, pH7.5, 200 mM NaCl, 1% Igepal ca-630, 10 mM MgCl2, and proteaseinhibitors). The lysate was cleared by centrifugation and thesupernatants were then flash-frozen and stored at −80° C. until testing.For the ELISA assay, HisGrab nickel coated 96-well plate strips (Pierce,#15142) were washed three times with ELISA buffer (200 μl consisting of50 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20, and 10 mM MgCl2). RalBP1(0.5 μg/100 μl) was then added to the wells and incubated with rocking(2 h RT). The plates were then washed three times with 200 μl ELISAbuffer. The plates were placed on ice and lysates, or lysis buffercontrol (100 μ1), were added to the wells in quadruplicate. The plateswere then incubated overnight with rocking at 4° C. followed by twowashes with ice-cold ELISA buffer. Mouse anti-FLAG (Sigma, F1804)antibody (1:20,000 in ELISA buffer) was then added at 100 μ1 per welland incubated (1 h, 4° C.). After three washes, goat anti-mouse antibodyconjugated to HRP (Pierce, #31430) (1:2,500) was added at 100 μ1 perwell and incubated (1 h, 4° C.). HRP substrate (Vector Laboratories,#SK-4400) was added to each well at 100 μl after three washes andincubated for 1 h at RT. The reactions were stopped by adding sulfuricacid (100 μl, 2N). Absorbance was read at OD450 on a Biotek Synergy H1plate reader (BioTek Instruments, Inc., Winooski, Vt.); Absorbance wascorrected for background absorbance by subtracting the reading for thesame well at OD540.

The J82 human bladder cancer cells stably expressing FLAG-tagged RalAimproved protein detection over that provided by anti-Ral antibodies.This afforded an enhanced dynamic range to the assay. The amount ofbound RalA was proportional to the relative activation state.

An independent approach was used to assess RalA activity, which isrequired for lipid raft exocytosis during spreading of murine embryonicfibroblasts (MEFs) on fibronectin-coated coverslips. Briefly, wild typeor caevolin-/- mouse embryonic fibroblasts were starved for 24 h,detached from culture plates with Accutase (Innovative Cell TechnologiesInc., San Diego, Calif.), resuspended in DMEM with 0.2% serum and 0.5%methyl cellulose, and held in suspension (90 min, 37° C.). While insuspension, cells were treated with inhibitor (50 μM or DMSO control, 1h). After treatment, cells were rinsed once with DMEM containing 0.2%serum and equal numbers of cells from all treatments were added to24-well plates that had been coated overnight (4° C., 2 μg/mL humanfibronectin). Cells were allowed to spread for 30 min and then fixedwith formaldehyde using standard protocols. To enable visualization,cells were labeled with Lava Cell (Active Motif) and visualized on aNikon TE300 fluorescence microscope. Three distinct regions of each wellwere imaged and cell spread area was quantitated using ImageJ (NIH).

In these cells, siRNA depletion of RalA inhibits spreading, whereascaveolin (Cavi)-/- MEFs are resistant to RalA depletion. siRNA againsthuman RalA and RalB or both were obtained from Dharmacon (Boulder, Co.)using published sequences.

Example 3—In Vitro Binding Assays

To confirm the direct binding of the compounds to the target, we usedTROSY 15N-HSQC (Transverse Relaxation-Optimized Heteronuclear SingleQuantum Coherence) NMR. RalB (Q72L mutant) in a pET16b (Novagen) plasmidwas a kind gift from Dr. Darerca Owen (Cambridge University). RalB waspurified with additional steps for loading with GDP or thenon-hydrolyzable form of GTP, GMPNPP (GNP, Sigma-Aldrich). Uniform¹³C¹⁵N-double labeled protein was produced in M9 media supplemented with15N-NH4Cl and 13C-glucose. Samples were prepared for NMR in 50 mM sodiumphosphate, pH 7.6, 100 mM NaCl and 1 mM MgCl2. All NMR experiments wererecorded on an Agilent 900 MHz system at 25° C. Resonance assignmentsfor the RalB-GNP complex were obtained from previously published studiesdeposited in Biological Magnetic Resonance Bank (BMRB, code: 15230).Chemical shift assignments of the RalB-GDP complex were obtainedindependently using HNCACB, CBCA(CO)NH and COCNH-TOCSY experiments. AllNMR data was processed using NMRPipe and analyzed using CCPNMR analysisprogram. Assignment were obtained by automated assignment using PINEfollowed by manual verification. ¹⁵N-HSQC experiments were used tomonitor amide shifts from the RalB protein (100 μM) following theaddition of compound reconstituted in deuterated DMSO. DMSOconcentrations in the final sample were 0.5% or 1%; control samples weremade with 0.5% or 1% deuterated DMSO and all samples containingcompounds were compared to their corresponding DMSO control.

Because only the NMR structure of RalB in complex with GNP has beensolved (PDB code 2KE5, BMRB entry 15230), we focused on this isoform.The ¹⁵N-HSQC NMR spectrum of RalB-GDP and RalB-GNP were first determinedand the chemical shift differences were analyzed. NMR spectra were thenrecorded in the presence of RBC8 or DMSO control. Binding of smallmolecules to the protein was monitored by the perturbation of ¹⁵N-HSQCprotein amide peaks. The ¹⁵N-HSQC spectrum of RalB-GDP (100 μM) in theabsence and presence of 100 μM RBC8 showed changes in peak position ofrepresentative residues located in the allosteric site. On the otherhand, RBC8 did not bind to RalB-GNP under the same conditions asindicated by minimal chemical shift changes on the NMR spectrum.

Based on all data including structural features, a series of RBC8derivatives was synthesized and tested for binding in vitro. We choseBQU57 and BQU85 for further evaluation because of superior performancecompared to RBC8 and drug-like properties (FIG. 3A, FIG. 2).

The synthesis schemes for compounds BQU57 and BQU85 are shown in FIG.2A.

A.6-amino-1,3-dimethyl-4-(4-(trifluoromethyl)phenyl)-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(BQU57): 4-(Trifluoromethyl)benzaldehyde (500 mg, 2.87 mmol),malononitrile (190 mg, 2.87 mmol) and triethylamine (400 μl mL, 2.87mmol) in ethanol (10 mL) was stirred for 1.0 min and then1H-Pyrazol-5(4H)-one (321 mg, 2.87 mmol) and added, capped and stirredat room temperature (22 hr) and then concentrated and purified bychromatography (SiO₂; 2% MeOH in methylene chloride) to afford BQU57(445 mg, 1.33 mmol, 46% yield) as a yellow solid. ¹H-NMR (400 MHz)DMSO-D₆: 7.28 (s, 4H), 7.10 (brs, 2H), 4.64 (s, 1H), 3.57 (s, 3H), 1.64(s, 3H); ¹³C-NMR (100 MHz) DMSO-D₆: 160.1, 147.6, 144.7, 143.9, 142.9,129.9, 122.3, 121.4, 120.6, 96.2, 58.2, 36.8, 33.9, 12.8.B.6-amino-1-methyl-3-phenyl-4-(4-(trifluoromethoxy)phenyl)-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (BQUO85): A mixture of the4-(trifluoromethoxy) benzaldehyde (0.327 g, 1.72 mmol, 1.0 equ.),malononitrile (0.114 g, 1.72 mmol, 1.0 equ.) and triethylamine (0.240mL, 1.72 mmol, 1.0 equ.) in ethanol (6.0 mL) was stirred for 10 min,followed by the addition of 1H-Pyrazol-5(4H)-one (0.300 g, 1.72 mmol).The reaction mixture was concentrated after 22 h and purified by columnchromatography on SiO₂ (2% methanol in dichloromethane) to giveBQU_03_85 (174 mg, 0.421 mmol, 25%) as yellow solid. ¹H-NMR (400 MHz)DMSO: 7.47-7.45 (d, 2H), 7.26-7.24 (d, 2H), 7.20-7.14 (m, 7H), 5.08 (s,1H), 3.76 (s, 3H); ¹³C-NMR (100 MHz) DMSO: 159.3, 147.4, 146.0, 144.7,144.1, 133.1, 129.8, 128.6, 128.0, 126.5, 121.7, 121.2, 120.4, 95.3,59.3, 37.4, 34.5.

A detailed NMR titration of the binding between BQU57 and RalB-GDP wascarried out. The NMR spectrum of RalB-GDP (100 μM) in the absence(black) and presence (magenta) of 100 μM BQU57 is shown in FIG. 3B.Representative residues that experience dose-dependent chemical shiftchanges are shown in FIG. 3C. A chemical shift change map with 100 μM ofBQU57 was generated (FIG. 3D) and most of the residues that exhibitedsignificant chemical shift changes (highlighted bars) were located tothe switch-II (aa 70-77) and helix α2 (aa 78-85) region. In the absenceof a crystal structure of RalB-GDP, a homology model was generated basedon the sequence similarity to RalA-GDP and the residues that experiencedchemical shift changes in the presence of drug was mapped onto thismodel (FIG. 3E). This shows that the majority of the chemical shiftchanges localize to the allosteric site, and confirm that BQU57 isbinding to the predicted site. Similar to RBC8, BQU57 (100 μM) did notbind to RalB-GNP (100 μM) as indicated by minimal chemical shift changeson the NMR spectrum (FIG. 2B).

Analysis of the NMR chemical shift titrations revealed that binding ofBQU57 was stoichiometric up to the apparent limiting solubility of thedrug (estimated as approx. 75 μM in control experiments without protein)(FIG. 2C). Consequently the binding of BQU57 to RalB-GDP was thendetermined using Isothermal Titration calorimetry (ITC). ITC experimentswere carried out using the MicroCal iTC200 system. Both protein and drugwere prepared in 50 mM sodium phosphate, pH 7.6, 100 mM NaCl, and 1 mMMgCl₂. Final DMSO concentration was adjusted to 1%. RalB-GDP protein(500 μM) were loaded into the syringe and titrated into drug (25 μM) orbuffer alone as control. All experiments were carried out at 25° C. ITCyielded a K_(D)=7.7±0.6 μM (FIG. 3F). This result was confirmed bySurface Plasma Resonance (SPR). SPR experiments were carried out usingthe Biacore 3000 system. Running buffer: PBS, pH 7.4, 1 μM GDP, 2 mMMgCl₂, 3% DMSO. Regeneration buffer: PBS, pH 7.4, 1 μM GDP, 2 mM MgCl₂.RalB protein was immobilized on CMS chip; samples of compound BQU57 inrunning buffer were injected at 30 μL/min for 60 s contact time followedby 5 minute regeneration. SPR gave a K_(D) of 4.7±1.5 μM despite lowsensitivity of the assay.

Differential scanning fluorometry (DSF) was used to evaluate bindingbetween compounds and RalB-GDP. The melting temperature was measured bymonitoring the increase of SYPRO orange that binds to hydrophobicregions of the protein. DSF was performed by preparing a platecontaining 10 μM RalB-GDP and 10 μM RalB-GPNPP, 4× SYPRO orange in 20 mMTris PH 8.0, 200 mM NaCl, 2.5 mM MgCl₂ and 1 mM DTT buffer. Testcompound was added to each well ensuring that the final concentration ofDMSO was 1% across all samples. The thermal melting curves were obtainedon a Light cycler 480 (Roche). The melting temperature was obtained bynormalizing the curves and obtaining the temperature at the midpoint ofthe transition curve. DSF confirmed dose-dependent binding between BQU57and RalB-GDP, and also demonstrated nucleotide-dependence.

Example 4—Effects on In Vitro Human Cancer Cell Growth

The effects of RBC8 and BQU57 on human lung cancer cell growth wereevaluated. Because Ral is well-known for its role in anchorageindependence, we carried out growth inhibition assays in soft agar. Fourhuman lung cancer cells H2122, H358, H460 and Calu6 were used in aseries of experiments to determine drug uptake, biologic specificity andeffect. To measure growth inhibition of human lung cancer cells underanchorage-independent conditions in soft agar, cells were seeded into6-well plates (coated with a base layer made of 2 ml of 1%low-melting-point agarose) at 15,000 cells per well in 3 ml of 0.4%low-melting-point agarose containing various concentration of drug. Twoto four weeks (depending on cell line) after incubation, cells werestained with 1 mg/ml MTT and colonies were counted under a microscope.The IC50 values were defined as the concentration of drug that resultedin 50% reduction in colony number compared to DMSO treated control.

For growth effects induced by siRNA treatment, cells were transfectedwith 50 nM siRNA against RalA, RalB or both (RalA/B) using methods andsequences described 10. After 72hrs, cells were subjected to the softagar colony formation assay.

For the chemo-genetic experiments, siRNA treated cells were seeded intosoft agar in the presence of various concentrations of drug. For theoverexpression experiments, H358 cells stably overexpressing FLAG,FLAG-RalAG23V or FLAG-RalBG23V were generated and cells were subjectedto the soft agar colony formation assay in the presence of drug.Attempts to stably overexpress FLAG-RalAG23V or FLAG-RalBG23V in H2122cells were unsuccessful and the rescue experiments with H2122 werecarried out 72 hrs after the transient transfection with FLAG,FLAG-RalAG23V or FLAG-RalBG23V using the soft agar colony formationassay in the presence of drug.

To quantitate how well the test compounds get into cells, H2122 humanlung cancer cells were seeded at 3×10⁵ cells per well in 6-well platesand let sit for 16 h. Compounds (10 μM) were individually dosed intriplicate; cells were then collected into 500 μl ice-cold ACN:MeOH:H₂0(1:1:1) at different time points (1, 5, 15, 30 and 60 min). Drugconcentrations in cell lysates were then determined using LC/MS-MSmethods as described with respect to the pharmacokinetic andpharmacodynamic studies in mice, described in detail in Example 5,below.

Testing cellular uptake of RBC8, BQU57, and BQU85 showed that all drugsreadily get into cells (FIG. 6). To confirm that Ral activity isinhibited in H2122 and H358 cells by drug treatment, we performed theRal activity pull-down assay. Cells were treated with drug for 3 hrs,collected and Ral activity measured using the RalBP1 pull-down assay kit(Millipore #14-415). RBC8 and BQU57 but not RBCS inhibited both RalA andRalB activity in both cell lines (FIG. 6E).

Additionally, all lines were found to be sensitive to K-Ras siRNAdepletion (FIG. 7A, 7B) but only H2122 and H358 were sensitive to Ralknockdown (FIG. 7C, 7D). Using this characteristic to determine thespecificity of the compounds to Ral compared to Ras, a closely relatedGTPase, we evaluated inhibition of colony formation in soft agar andnoted the Ral-dependent lines H2122 and H358 but not in H460 or Calu6cells were sensitive (FIG. 4A, B, K). The IC50 for RBC8 is 3.5 μM inH2122 and 3.4 μM in H358; for BQU57 2.0 μM in H2122 and 1.3 μM in H358.Next a chemo-genomic experiment was performed to further determine drugspecificity to Ral. Treatment of H2122 and H358 cells that had siRNAknockdown of RalA and RalB with RBC8 or BQU57 did not result insignificant further inhibition (FIGS. 4C-4F, FIG. 7E). Together, thisdata suggest RBC8, BQU57, and BQU85 reduce anchorage independent growthvia Ral inhibition.

To address the specificity of the compounds for the GDP compared to theGTP form of Ral, we overexpressed constitutively active forms ofRalAG23V or RalBG23V in H2122 and H358 cells. The G23V mutation preventsRalGAP mediated activation of GTP hydrolysis and hence locks Ral in itsactive state 30. We found that both RalAG23V and RalBG23V could rescuethe growth inhibition effect of the compounds (FIGS. 4G-4J, FIG. 7F).

Example 5—Pharmacokinetics, Pharmacodynamics and Tumor Growth In Vivo

Inhibition of Ral activity and tumor growth were evaluated in human lungcancer mouse models. Pharmacokinetics (PK) of RBC8 and BQU57 were firstanalyzed in nude mice to test bioavailability. Following a singleintraperitoneal injection (50 mg/Kg), blood samples were collected attime intervals from 0 to 5 h post-dose (9 time points). Pharmacokineticparameters including area under the curve (AUC), Cmax, and t½ wereestimated using non-compartmental methods by LC-MS/MS and showedfavorable properties that define good drug candidates (see Table 1,supra).

We next determined compound entry into tumor tissue. To do so, athymicnude mice (Ncr nu/nu; National Cancer Institute, Fredrick, Md.) werereceived at 5 to 6 weeks of age and were allowed to acclimate for 2weeks in sterile micro isolator cages with constant temperature andhumidity. Mice had free access to food and water. H2122 cells inlog-phase growth were harvested on the day of use. Cells were suspendedin un-supplemented RPMI 1640 medium and 0.1 mL (2×10⁵ cells) wasinjected s.c. four sites per mice. For H358 xenografts, cells (5×10⁶)were mixed with matrigel (20% final concentration) and 0.1 mL wasinoculated s.c. per site. After cell inoculation, mice were monitoreddaily, weighed twice weekly and caliper measurements begun when tumorsvisible. Tumor volume was calculated by (L×W2)/2, where L is longermeasurement of tumor and W is the smaller tumor measurement. Drugtreatment started the day after inoculation. Compounds were dissolved inDMSO and injected i.p. daily except weekends at 10/20/50 mg/kg. Noobvious toxicities were observed in the control (DMSO) or drug-treatedanimals as assessed by difference in body weight between control anddrug-treated animals taking tumor size into account. As shown in FIG.5A, B, G substantial amounts of compound were detected in tumor tissue 3h post-dose. The effect of the Ral inhibitors on xenograft tumor growthwas then tested in nude mice. Mice were inoculated subcutaneously withH2122 human lung cancer cells and treated intraperitoneally with 50mg/kg/d (except weekends) of RBC8 24 h post inoculation. RBC8 inhibitedtumor growth (FIG. 5C-D) by the same order of magnitude as dualknockdown of RalA and RalB (FIG. 5E), and a second lung cancer line,H358 yielded similar results. BQU57 and BQU85 were also tested in vivoat several different doses (5, 10, 20, and 50 mg/kg/d) anddose-dependent growth inhibition effects were observed (FIGS. 5F,5H).

Finally, we evaluated Ral GTPase activity in vivo in the H2122xenografts. Nude mice were inoculated with 5×10⁶ cells H2122 cells s.c.at four sites per mice. Tumor size reached an average of 250 mm³ in tendays, at which time mice were given a signal i.p. dose of RBC8 or BQU57at various concentrations. Tumors were then collected 3 h afterinjection of RBC8 or BQU57. RalA and RalB activity in tumor samples werethen measured using the RalBP1 pull-down assay kit (Millipore #14-415).Ras and RhoA activity in tumor samples were measured using therespective pull-down assay kits. All the activity assays used westernblotting as the final readout. For quantification of the immunoblots,the bands on each blot were first normalized to their respectiveinternal control (10 ng of recombinant Ral, Ras, or Ral protein run inthe last lane) the numbers were then compared across different blots,each of which represented one treatment condition. Mice bearing H2122tumors (median size 250 mm³) were given a single intraperitoneal dose ofRBC8 (50 mg/kg) or BQU57 (10/20/50 mg/kg) and tumors collected 3 hpost-dose. RalBP1 pull-down measurements of Ral activity showedsignificant inhibition of both RalA and RalB by RBC8 and BQU57.Importantly, BQU57-induced dose-dependent inhibition of Ral activitycorrelated with inhibition of tumor growth, and Ras and RhoA activitywas also measured in BQU57 treated tumors and no significant inhibitionwas observed, further demonstrating the selectivity of these Ralinhibitors.

Example 6—Synthesis of Compounds of the Invention

Compounds of the invention were synthesized according to the followingsynthesis scheme and materials.

Compound Synthesis Scheme

Compound Number R1 R2 R3 R4 R5 R6 R7 1 Ph Me — — — — — 2 Ph Me — — — — —3 Me Ph — — — — — 4 Ph Ph — — — — — CH₂— 5 Ph Ph — — — — — m,p-DiOMe— 6Me Ph — — — — — m,p-DiOMe— 7 Ph Ph — — — — — 8 Ph p-OMe—Ph — — — — — 9Me p-OMe—Ph — — — — — 10 Me Me H H H H H 11 Me Ph H H H H H 12 Ph Me H HH H H 13 Ph Ph H H H H H 14 Ph p-OMe—Ph H H H H H 15 Me p-OMe—Ph H H H HH m,p-diOMe— 16 Me Ph H H H H H 17 Me Me F H H H H 18 Ph Me F H H H H 19Me Ph F H H H H 20 Ph Ph F H H H H 21 Me m,p-diOMe— F H H H H Ph 22 Php-OMe—Ph F H H H H m,p-diOMe— 23 Ph Ph F H H H H 24 Me p-OMe—Ph F H H HH 25 Me Me H F H H F 26 Me Ph H F H H F 27 Ph Me OMe H H H H 28 Me MeOMe H H H H 29 Me Ph OMe H H H H 30 Ph Ph OMe H H H H 31 Ph p-OMe—Ph OMeH H H H 32 Me p-OMe—Ph OMe H H H H 33 Me Me OMe F H H H 34 Me Me OMe HOMe OMe H 35 Me Ph OMe F H H H 36 Me Me CF₃ H H H H 37 Me Ph CF₃ H H H H38 Me Me O—CF₃ H H H H 39 Me Ph O—CF₃ H H H H 40 Me Me CN H H H H 41 MePh CN H H H H 42 Me Me CH(CH3)₂ H H H H 43 Me Ph CH(CH₃)₂ H H H H CH₂—44 Me Me O—CH₂— O— H H H CH₂— 45 Me Ph O—CH₂— O— H H H 46 Me Me N: H H HH 47 Me Ph N: H H H H 48 Me Me H N: H H H 49 Me Ph H N: H H H 50 Me Me HBr N: H H 51 Me Ph H Br N: H H 52 Me Me imidazole H H H H 53 Me Phimidazole H H H H

Materials and Methods

Anisaldehyde, benzaldehyde, 1,4-benzodioxan-6-carboxaldehyde,benzyl-hydrazine, 6-bromo-2-pyridincarboxaldehyde, deuterated chloroform(CDCl₃), deuterated dimethyl sulfoxide (DMSO-d6),3,5-difluorobenzaldehyde, ethyl acetoacetate, ethyl benzoylacetate,ethyl 3,4-dimethoxybenzoylacetate, ethyl-hydrocupreine hydrochloride,ethyl-4-methoxybenzoylacetate, 4-fluorobenzaldehyde,3-fluoro-4-methoxybenzaldehyde, 4-formylbenzonitrile,4-isopropylbenzaldehyde, 4-(1H-imidazol-1-yl)benzaldehyde,malononitrile, methyl-hydrazine, phenyl-hydrazine,3-pyridincarboxaldehyde, sodium ethoxide, trimethylamine (TEA),2,4,6-trimethoxybenzaldehyde, and 4-(trifluoromethoxy) benzaldehyde werepurchased from Sigma-Aldrich Chemical Company (St. Louis, Mo.). Ethylacetate (EtOAc), HPLC grade methanol (MeOH), HPLC grade acetonitrile(ACN), HPLC grade water (H₂O), formic acid, ammonium acetate, hexanesand methylene chloride (DCM) were obtained from Fisher Scientific(Pittsburgh, Pa.). Ethanol was purchased from Decon Laboratories, Inc.(King of Prussia, Pa.). Silica Gel 60 Å 40-63 μm was purchased fromSorbent Technologies (Norcross, Ga.).

The ¹H and ¹³C NMR spectra were recorded using a 400 MHz Bruker NMR,Avance III 400. The chemical shifts are reported in ppm. An AppliedBiosystems Sciex 4000 (Applied Biosystems; Foster City, Calif.) whichwas equipped with a Shimadzu HPLC (Shimadzu Scientific Instruments,Inc.; Columbia, Md.) and Leap auto-sampler (LEAP Technologies; Carrboro,N.C.) was used. Liquid chromatography employed an Agilent Technologies,Zorbax extended-C18 50×4.6 mm, 5 micron column at 40° C. with aflow-rate of 0.4 mL/min. The mobile phase consisted of A: 10 mM(NH₄OAc), 0.1% formic acid in H₂O, and B: 50:50 ACN:MeOH. Thechromatography method used was 95% A for 1.0 min; ramped to 95% B at 3.0min and held for 4.5 min, lastly, brought back to 95% A at 8.5 min andheld for 1.0 min (9.5 min total run time). Synthesized compounds weremonitored via electro-spray ionization positive ion mode (ESI+) usingthe following conditions: i) an ion-spray voltage of 5500 V; ii)temperature, 450° C.; iii) curtain gas (CUR; set at 10) andCollisionally Activated Dissociation (CAD; set at 5) gas were nitrogen;iv) Ion Source gas one (GS1) and two (GS2); v) entrance potential wasset at 10 V; vi) quadruple one (Q1) and (Q3) were set on Unitresolution; vii) dwell time was set at 200 msec; and viii) declusteringpotential (DP), collision energy (CE), and collision cell exit potential(OCP) are voltages (V). Samples (10 μL) were analyzed by LC/MS-MS. Asjudged by NMR and LC/MS-MS analysis, all purified compounds were >97%pure.

Synthesis:

3-methyl-1-phenyl-1H-pyrazol-5(4H)-one (1): A solution of ethylacetoacetate (9.02 mL, 71.2 mmol) in EtOH (130 mL) was treated at 0° C.with phenyl-hydrazine (7.00 g, 64.7 mmol). The mixture was allowed toslowly warm to ambient temperature and then heated to 60° C. (3 h). Thesolvent was removed under vacuum and the residue purified by columnchromatography on silica gel (EtOAc:hexanes; 1:1) to give 1 (7.60 g,43.6 mmol, 67% yield) as a light yellow powder. ¹H-NMR (400 MHz) CDCl₃:7.87-7.85 (d, 2H), 7.41-7.37 (t, 2H), 7.19-7.16 (t, 1H), 3.42 (s, 2H),2.19 (s, 3H), ¹³C-NMR (100 MHz) CDCl₃: 170.5, 156.2, 138.0, 128.8,125.0, 118.8, 43.0, 17.0; LC/MS-MS: 175.0→77.1 m/z; GS1 and GS2 at 30,DP=56, CE=25, CXP=4, t_(R)=3.52 min.1,3-dimethyl-1H-pyrazol-5(4H)-one (2): Ethyl acetoacetate (15.1 mL, 119mmol) in EtOH (200 mL) was treated at 0° C. with methyl-hydrazine (5.00g, 109 mmol). The mixture was allowed to slowly warm to ambienttemperature and then heated to 60° C. (3 h). The solvent was removedunder vacuum and the residue purified by column chromatography on silicagel (EtOAc:hexanes; 1:1) to afford 2 (8.02 g, 71.5 mmol, 66% yield)after purification by crystallization (DCM and hexanes) as a white offsolid. ¹H-NMR (400 MHz) CDCl₃: 3.25 (s, 3H), 3.16 (s, 2H), 2.08 (s, 3H),¹³C-NMR (100 MHz) CDCl₃: 172.2, 155.4, 138.0, 41.3, 31.0, 16.8.LC/MS-MS: 113.2→82.0 m/z; GS1 and GS2 at 30, DP=61, CE=25, CXP=4,t_(R)=2.9 min.1-methyl-3-phenyl-1H-pyrazol-5(4H)-one (3): Ethyl benzoylacetate (18.4mL, 95.5 mmol) in EtOH (180 mL) was treated at 0° C. withmethyl-hydrazine (4.57 mL, 86.8 mmol.). The mixture was allowed toslowly warm to ambient temperature and then heated to 60° C. (3 h). Thesolvent was removed under vacuum and the residue purified by columnchromatography on silica gel (EtOAc:hexanes; 1:1) to give 3 (11.0 g 63.1mmol, 73% yield) after purification by crystallization (ethanol) as alight yellow solid. ¹H-NMR (400 MHz) CDCl₃: 7.67-7.65 (m, 2H), 7.42-7.41(m, 3H), 3.60 (s, 2H), 3.41 (s, 3H), ¹³C-NMR (100 MHz) CDCl₃: 171.8,154.2, 131.0, 130.3, 128.8, 125.6, 37.9, 31.4. LC/MS-MS: 175.0→77.2 m/z;GS1 and GS2 at 30, DP=66, CE=43, CXP=4, t_(R)=3.45 min.1,3-diphenyl-1H-pyrazol-5(4H)-one (4): Ethyl benzoylacetate (12.2 mL,71.2 mmol) in EtOH (130 mL) was treated at 0° C. with phenyl-hydrazine(7.00 g, 71.2 mmol.). The mixture was allowed to slowly warm to ambienttemperature and heated to 60° C. (3 h). The solvent was removed undervacuum and the residue purified by column chromatography on silica gel(EtOAc:hexanes; 1:4) and crystallization (EtOH) to give 4 as anoff-white solid (6.75 g, 28.6 mmol, 44% yield). ¹H-NMR (400 MHz) DMSO:11.8 (s, 1H), 7.84-7.82 (d, 4H), 7.50-7.40 (m, 4H), 7.34-7.27 (m, 2H),6.02 (s, 1H), ¹³C-NMR (100 MHz) DMSO: 154.2, 150.0, 139.3, 133.8, 129.3,129.0, 128.2, 126.1, 125.5, 121.5, 85.5; LC/MS-MS: 237.0→77.1 m/z; GS1and GS2 at 30, DP=81, CE=68, CXP=4, t_(R)=4.15 min.1-benzyl-3-phenyl-1H-pyrazol-5(4H)-one (5): A solution of ethylbenzoylacetate (4.80 mL, 28.2 mmol) in EtOH (60 mL) was treated at 0° C.with benzyl-hydrazine (5.00 g, 25.6 mmol). The mixture was slowly warmedto ambient temperature and heated to 60° C. (16 h). The reaction mixturewas concentrated and diluted with EtOH (100 mL) and then 3.0 g of sodiumethoxide added and stirred (40 h). The solid was filtered off and thesolvent removed in vacuo. The residue was purified by columnchromatography on silica gel (4:1 hexanes:EtOAc to 100% EtOAc) to give 5(25.5 mg, 1.02 mmol, 4% yield) as a light orange solid. ¹H-NMR (400 MHz)DMSO: 11.2 (s, 1H), 7.71-7.70 (d, 2H), 7.37-7.31 (m, 4H), 7.27-7.20 (m,4H), 5.85 (s, 1H), 5.13 (s, 2H), ¹³C-NMR (100 MHz) DMSO: 153.6, 148.6,138.3, 134.4, 128.8, 128.7, 127.6, 127.5, 125.1, 83.7, 50.0; LC/MS-MS:251.1→91.1 m/z; GS1 and GS2 at 30, DP=2, CE=33, CXP=14, t_(R)=4.01 min.3-(3,4-dimethoxyphenyl)-1-methyl-1H-pyrazol-5(4H)-one (6): Ethyl3,4-dimethoxybenzoylacetate (5.00 g, 19.8 mmol) in EtOH (60 mL) wastreated at 0° C. with methyl-hydrazine (0.95 mL,19.8 mmol, 1.0 equiv.).The mixture was allowed to slowly warm to ambient temperature and heatedto 60° C. (3 h). The solvent was removed under vacuum and the residuepurified by chromatography on silica gel (hexanes:EtOAc; 4:1 to 1:1) to6 (1.86 g, 7.94 mmol, 44% yield) as a light yellow powder. ¹H-NMR (400MHz) CDCl₃: 7.35-7.35 (d, 1H), 7.06-7.04 (dd, 1H), 6.87-6.85 (d, 1H),3.94 (s, 3H), 3.92 (s, 3H), 3.57 (s, 2H), 3.39 (s, 3H); ¹³C-NMR (100MHz) CDCl₃: 171.6, 154.1, 151.1, 149.4, 124.1, 119.6, 110.7, 107.3,55.9, 55.9, 38.0, 31.3; LC/MS-MS: 235.1→219.0 m/z; GS1 and GS2 at 30,DP=66, CE=33, CXP=14, t_(R)=3.26 min.3-(3,4-dimethoxyphenyl)-1-phenyl-1H-pyrazol-5(4H)-one: (7): Ethyl3,4-dimethoxybenzoylacetate (3.00 g, 11.9 mmol.) in EtOH (60 mL) wastreated at 0° C. with phenyl-hydrazine (1.17 mL, 10.8 mmol.). Themixture was allowed to slowly warm to ambient temperature and heated to60° C. (3 h). The solvent was removed under vacuum and the residuepurified by chromatography on silica gel (hexanes:EtOAc; 4:1 to 1:1) toafford 7 (920 mg, 2.32 mmol, 22% yield) after purification bycrystallization (EtOH) as a yellow powder. ¹H-NMR (400 MHz) CDCl₃:8.00-7.97 (d, 1H), 7.48-7.42 (m, 3H), 7.25-7.21 (t, 1H), 7.17-7.14 (dd,1 H), 6.91-6.89 (d, 1H), 3.98 (s, 3H), 3.95 (s, 3H), 3.83 (s, 2H);¹³C-NMR (100 MHz) CDCl₃: 170.1, 154.4, 151.4, 149.4, 138.1, 128.8,125.2, 123.8, 120.1, 119.1, 110.7, 107.6, 56.0, 56.0, 39.7; LC/MS-MS:297.0→218.2 m/z; GS1 and GS2 at 30, DP=96, CE=37, CXP=18, t_(R)=3.98min.3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-5(4H)-one (8):Ethyl-4-methoxybenzoylacetate (7.00 g, 27.8 mmol) in EtOH (100 mL) wastreated at 0° C. with phenyl-hydrazine (2.50 mL, 25.3 mmol). The mixturewas allowed to slowly warm to ambient temperature and heated to 60° C.(3 h). The solvent was removed under vacuum and the residue purified bychromatography on silica gel (hexanes:EtOAc; 4:1 to 1:1) to afford3-(4-methoxyphenyl) -1-phenyl-1H-pyrazol-5(4H)-one (8; 5.21 g, 19.6mmol, 78% yield) after crystallization (EtOH) as a light yellow solid.¹H-NMR (400 MHz) CDCl₃: 7.99-7.97 (d, 1H), 7.66-7.64 (d, 2H), 7.44-7.40(t, 2H), 7.22-7.18 (t, 1H), 6.94-6.92 (d, 2H), 3.82 (s, 3H), 3.68 (s,3H); ¹³C-NMR (100 MHz) CDCl₃: 170.1, 161.5, 154.4, 138.2, 128.8, 127.5,125.0, 123.5, 118.8, 114.2, 55.3, 39.6; LC/MS-MS: 267.0→77.2 m/z; GS1and GS2 at 30, DP=81, CE=65, CXP=4, t_(R)=4.15 min.3-(4-methoxyphenyl)-1-methyl-1H-pyrazol-5(4H)-one (9):Ethyl-4-methoxybenzoylacetate (7.00 g, 27.8 mmol) in EtOH (100 mL) wastreated at 0° C. with methyl-hydrazine (1.30 mL, 25.2 mmol). The mixturewas allowed to slowly warm to ambient temperature and heated to 60° C.(3 h). The solvent was removed under vacuum and the residue purified bychromatography on silica gel (hexanes:EtOAc; 4:1 to 1:1) to afford 9(3.00 g, 14.7 mmol, 58% yield) after crystallization from EtOH as alight yellow solid. ¹H-NMR (400 MHz) DMSO: 10.9 (s, 1H), 7.63-7.60 (d,2H), 6.92-6.90 (d, 2H), 5.70 (s, 1H), 3.76 (s, 3H), 3.54 (s, 3H);¹³C-NMR (100 MHz) CDCl₃: 161.1, 153.4, 147.9, 126.3, 114.6, 114.4, 83.1,59.7, 31.3; LC/MS-MS: 205.0→190.1 m/z; GS1 and GS2 at 30, DP=51, CE=29,CXP=12, t_(R)=3.44 min.6-amino-1,3-dimethyl-4-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(10): A mixture of benzaldehyde (290 μL, 2.87 mmol), malononitrile (190mg, 2.87 mmol) and TEA (400 μL, 2.87 mmol) in EtOH (10 mL) was stirredfor 1.0 min, followed by the addition of 2 (322 mg, 2.87 mmol). Thereaction mixture was concentrated after 19 h and washed with EtOH andhexanes. The crude material was purified by column chromatography onSiO₂ (25% EtOAc in hexanes ramped to 100% EtOAc) and thenre-crystallized from EtOH to give 10 (263 mg, 0.988 mmol, 34% yield) asa yellow powder. ¹H-NMR (400 MHz) DMSO: 7.34-7.32 (m, 2H), 7.25-7.23 (t,1H), 7.19-7.17 (d, 2H), 7.05 (s, 2H), 4.57 (s, 1H), 3.60 (s, 3H), 1.66(s, 3H); ¹³C-NMR (100 MHz) DMSO: 159.9, 144.6, 144.4, 142.9, 128.8,128.0, 127.3, 120.6, 96.5, 58.7, 37.5, 33.8, 12.8; LC/MS-MS: 267.0→201.3m/z; GS1 and GS2 at 30, DP=61, CE=29, CXP=12, t_(R)=3.74 min.6-amino-1-methyl-3,4-diphenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(11): A mixture consisting of benzaldehyde (290 μL, 2.87 mmol),malononitrile (190 mg, 2.87 mmol,) and TEA (400 μL, 2.87 mmol) in EtOH(10 mL) was stirred for 1.0 min, followed by the addition of 3 (500 mg,2.87 mmol). The reaction mixture was concentrated after 21 h and washedwith EtOH and hexanes; re-crystallized from EtOH to give 11 (282 mg,8.58 mmol, 30% yield) as a white solid. ¹H-NMR (400 MHz) DMSO: 7.41-7.38(m, 2H), 7.28-7.24 (m, 2H), 7.21-7.18 (m, 6H), 4.88 (s, 1H), 4.77 (s, 2H), 3.83 (s, 3H); ¹³C-NMR (100 MHz) DMSO: 158.1, 146.0, 144.8, 144.6,133.2, 128.7, 128.5, 127.9, 127.8, 127.1, 126.4, 120.5, 95.7, 59.9,38.2, 34.5; LC/MS-MS: 329.1→263.1 m/z; GS1 and GS2 at 30, DP=71, CE=31,CXP=18, t_(R)=4.00 min.6-amino-3-methyl-1,4-diphenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(12): To a stirred solution of benzaldehyde (290 μL, 2.87 mmol),malononitrile (190 mg, 2.87 mmol) and 1 (500 mg, 2.87 mmol) in anhydrousDCM (60 mL) was added anhydrous Na₂SO₄ (407 mg, 2.87 mmol) andethyl-hydrocupreine hydrochloride (46 mg, 0.122 mmol). The reactionmixture was stirred at room temperature (25 h). After filtration andwashing with DCM, the solvent was removed under reduced pressure. Thecrude mixture was subjected to flash column chromatography over silicagel (hexanes:EtOAc; 1:1) to give 12 (270 mg, 0.822 mmol, 29% yield) as awhite solid. ¹H-NMR (400 MHz) CDCl₃: 7.69-7.66 (d, 2H), 7.50-7.46 (t,2H), 7.39-7.26 (m, 6H), 4.68 (s, 1H), 4.67 (s, 2H), 1.91 (s, 3H),¹³C-NMR (100 MHz) CDCl₃: 158.1, 146.4, 143.8, 141.9, 137.5, 129.2,128.8, 127.8, 127.5, 126.7, 121.2, 119.0, 98.3, 64.0, 37.4, 12.8;LC/MS-MS: 329.1→263.1 m/z; GS1 and GS2 at 30, DP=56, CE=31, CXP=18,t_(R)=4.18 min.6-amino-1,3,4-triphenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(13): A mixture of benzaldehyde (290 μL, 2.87 mmol), malononitrile (190mg, 2.87 mmol) and TEA (400 μL, 2.87 mmol) in EtOH(10 mL) was stirredfor 1.0 min, followed by the addition of 4 (678 mg, 2.87 mmol). Theprecipitate was filtered off and washed with EtOH and hexanes, andre-crystallized from EtOH to give 13 (330 mg, 0.845 mmol, 29% yield) asa white solid. ¹H-NMR (400 MHz) CDCl₃: 7.82-7.80 (d, 2H), 7.55-7.50 (m,4H), 7.41-7.37 (t, 1H), 7.32-7.22 (m, 8H), 4.96 (s, 1H), 4.68 (s, 2H);¹³C-NMR (100 MHz) CDCl₃: 157.5, 147.7, 144.9, 142.6, 137.5, 132.2,129.3, 128.8, 128.2, 128.1, 127.5, 127.4, 127.1, 126.9, 121.6, 118.9,97.5, 64.8, 38.2; LC/MS-MS: 391.1→325.0 m/z; GS1 and GS2 at 30, DP=91,CE=33, CXP=22, t_(R)=4.33 min.6-amino-3-(4-methoxyphenyl)-1,4-diphenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(14): A mixture of benzaldehyde (290 μL, 2.87 mmol, 1.0 equ.),malononitrile (190 mg, 2.87 mmol, 1.0 equ.) and TEA (400 μL, 2.87 mmol,1.0 equ.) in EtOH (10 mL) was stirred for 1.0 min, followed by theaddition of 8 (764 mg, 2.87 mmol). The reaction mixture was concentratedafter 19 h and washed with EtOH and hexanes, re-crystallized from EtOHto give 14 (695 mg, 1.65 mmol, 58% yield) as a white solid. ¹H-NMR (400MHz) DMSO: 7.94-7.92 (d, 2H), 7.58-7.53 (m, 4H), 7.41-7.37 (t, 1H),7.27-7.16 (m, 7H), 6.83-6.81 (d, 2H), 5.04 (s, 1H), 3.71 (s, 3H);¹³C-NMR (100 MHz) DMSO: 159.5, 159.0, 146.6, 145.6, 144.5, 137.9, 129.8,128.9, 128.3, 128.0, 127.3, 127.1, 125.1, 121.1, 120.3, 114.1, 97.5,59.8, 55.5, 37.9; LC/MS-MS: 421.2→355.0 m/z; GS1 and GS2 at 30, DP=71,CE=33, CXP=24, t_(R)=4.3 min.6-amino-3-(4-methoxyphenyl)-1-methyl-4-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(15): A mixture of benzaldehyde (290 μL, 2.87 mmol, 1.0 equ.),malononitrile (190 mg, 2.87 mmol, 1.0 equ.) and TEA (400 μL, 2.87 mmol,1.0 equ.) in EtOH (10 mL) was stirred for 1.0 min, followed by theaddition of 9 (583 mg, 2.87 mmol). The reaction mixture was concentratedafter 24 h. The crude material was purified by column chromatography(25% EtOAc in hexanes and ramped to 100% EtOAc), then re-crystallizedfrom EtOH to give 15 (80.9 mg, 8% yield, 0.226 mmol) as a yellow solid.¹H-NMR (400 MHz) DMSO: 7.42-7.40 (d, 2H), 7.23-7.21 (m, 2H), 7.15-7.13(d, 3H), 7.06 (s, 2H), 6.77-6.75 (d, 2H), 4.93 (s, 1H), 3.76 (s, 3H),3.69 (s, 3H); ¹³C-NMR (100 MHz) DMSO: 159.1, 159.0, 145.9, 144.8, 144.5,128.8, 127.8, 127.7, 127.1, 125.8, 120.5, 113.9, 95.0, 59.9, 55.4, 38.2,34.4; LC/MS-MS: 359.1→293.0 m/z; GS1 and GS2 at 30, DP=76, CE=31,CXP=20, t_(R)=4.0 min.6-amino-3-(3,4-dimethoxyphenyl)-1-methyl-4-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(16): A mixture of benzaldehyde (145 μL, 1.44 mmol), malononitrile (90.0mg, 1.44 mmol) and TEA (200 μL, 1.44 mmol) in EtOH (5.0 mL) was stirredfor 1.0 min, followed by the addition of 6 (336 mg, 1.44 mmol). Thereaction mixture was concentrated after 24 h. The crude material waspurified by column chromatography (25% EtOAc in hexanes ramped to 100%EtOAc), then re-crystallized from EtOH to give 16 (48.5 mg, 9% yield,0.124 mmol) as a yellow solid. ¹H-NMR (400 MHz) CDCl₃: 7.29-7.28 (d,2H), 7.23-7.21 (d, 2H), 7.00-6.98 (d, 1H), 6.88 (s, 1H), 6.72-6.70 (d,2H), 4.84 (s, 1H), 4.75 (s, 2H), 3.82 (s, 6H), 3.60 (s, 3H); ¹³C-NMR(100 MHz) CDCl₃: 157.6, 148.7, 148.6, 146.1, 145.7, 143.1, 128.9, 127.5,127.4, 125.6, 119.3, 119.2, 110.9, 109.7, 94.7, 64.4, 55.7, 55.6, 38.3,34.1; LC/MS-MS: 389.1→323.0 m/z; GS1 and GS2 at 30, DP=66, CE=31,CXP=22, t_(R)=3.82 min.6-amino-4-(4-fluorophenyl)-1,3-dimethyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(17): A mixture of 4-fluorobenzaldehyde (300 μL, 2.87 mmol),malononitrile (190 mg, 2.87 mmol) and TEA (400 mL, 2.87 mmol) in EtOH(8.0 mL) was stirred for 1.0 min, followed by the addition of 2 (322 mg,2.87 mmol). The reaction mixture was concentrated after 24 h and washedwith EtOH and hexanes, and re-crystallized from EtOH to give 17 (335 mg,41% yield, 1.17 mmol) as a white solid. ¹H-NMR (400 MHz) DMSO: 7.23-7.20(m, 2H), 7.16-7.12 (m, 2H), 7.07 (s, 2H), 4.61 (s, 1H), 3.60 (s, 3H),1.67 (s, 3H); ¹³C-NMR (100 MHz) DMSO: 162.7, 159.9 (d), 144.6, 142.9,140.7, 129.9, 120.6, 115.5 (d), 96.3, 56.4, 36.7, 33.8, 12.8; LC/MS-MS:285.1→219.1 m/z; GS1 and GS2 at 30, DP=61, CE=27, CXP=14, t_(R)=3.8 min.6-amino-4-(4-fluorophenyl)-3-methyl-1-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(18): A mixture of the 4-fluourobenzaldehyde (356 mg, 2.87 mmol),malononitrile (190 mg, 2.87 mmol, 1.0 equ.) and TEA (400 μL, 2.87 mmol)in EtOH (10 mL) was stirred for 1.0 min, followed by the addition of the1 (500 mg, 2.87 mmol). The reaction mixture was concentrated after 18 hand the precipitate filtered and re-crystallized from EtOH to give 18(85.0 mg, 0.245 mmol, 9% yield) as a white solid. ¹H-NMR (400 MHz)CDCl₃: 7.68-7.66 (d, 2H), 7.50-7.46 (t, 2H), 7.34-7.32 (t, 1H),7.28-7.22 (m, 2H) 7.08-7.04 (t, 2H), 4.68 (s, 3H), 1.91 (s, 3H); ¹³C-NMR(100 MHz) CDCl₃: 158.0, 146.2, 143.7, 137.8, 137.5, 129.4, 129.2, 126.8,121.2, 118.8, 115.8, 115.6, 98.1, 63.8, 36.7, 12.8; LC/MS-MS:347.1→281.1 m/z; GS1 and GS2 at 30, DP=11, CE=31, CXP=18, t_(R)=4.16min.6-amino-4-(4-fluorophenyl)-1-methyl-3-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(19): A mixture of the 4-fluorobenzaldehyde (300 μL, 2.87 mmol),malononitrile (190 mg, 2.87 mmol) and TEA (400 μL, 2.87 mmol) in EtOH(10 mL) was stirred for 1.0 min, followed by the addition of 3 (500 mg,2.87 mmol). The reaction mixture was concentrated after 20 h in vacuoand washed with EtOH and hexanes, and re-crystallized from EtOH to give19 (182 mg, 0.525 mmol, 18% yield) as a light yellow solid. ¹H-NMR (400MHz) DMSO: 7.50-7.48 (d, 2H), 7.22-7.18 (m, 5H), 7.11 (s, 2H), 7.05-6.98(t, 2H) 5.04 (s, 1H), 3.78 (s, 3H); ¹³C-NMR (100 MHz) DMSO: 162.5,159.1, 146.0, 144.6, 140.9, 133.1, 129.8 (d), 128.5, 127.9, 126.5,120.4, 115.4 (d), 95.5, 59.7, 37.4, 34.5; LC/MS-MS: 347.1→281.0 m/z; GS1and GS2 at 30, DP=66, CE=31, CXP=14, t_(R)=4.0 min.6-amino-4-(4-fluorophenyl)-1,3-diphenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(20): A mixture of 4-fluorobenzaldehyde (300 μL, 2.87 mmol),malononitrile (190 mg, 2.87 mmol) and TEA (400 μL, 2.87 mmol) in ethanol(10 mL) was stirred for 1.0 min, followed by the addition of 4 (678 mg,2.87 mmol). After 18 h, the precipitate formed was filtered out andwashed with EtOH and hexanes, and re-crystallized from EtOH to afford 20(240 mg, 0.588 mmol, 20% yield) as a white powder. ¹H-NMR (400 MHz)DMSO: 7.94-7.92 (d, 2H), 7.61-7.55 (m, 4H), 7.42-7.38 (t, 1H), 7.28-7.24(m, 7H), 7.06-7.02 (t, 2H), 5.15 (s, 1H), ¹³C-NMR (100 MHz) DMSO: 162.6,159.0, 146.8, 145.6, 140.6, 137.8, 132.5, 130.0, 129.9 (d), 128.6,128.6, 127.3, 127.0, 121.3, 120.2, 115.5 (d), 97.9, 59.6, 37.0;LC/MS-MS: 410.4→242.2 m/z; GS1 and GS2 at 30, DP=21, CE=47, CXP=16,t_(R)=4.6 min.6-amino-3-(3,4-dimethoxyphenyl)-4-(4-fluorophenyl)-1-methyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (21): A mixture of 4-fluorobenzaldehyde (300μL, 2.87 mmol), malononitrile (190 mg, 2.87 mmol, 1.0 equ.) and TEA (400μL, 2.87 mmol) in EtOH (10 mL) was stirred for 1.0 min, followed by theaddition of 6 (672 mg, 2.87 mmol). After 17 h, the precipitate formedwas filtered out and washed with EtOH and hexanes, and re-crystallizedfrom EtOH to give 21 (782 mg, 1.93 mmol, 67% yield) as a white powder.¹H-NMR (400 MHz) DMSO: 7.20-7.18 (m, 2H), 7.09-7.03 (m, 5H), 6.96-6.95(d, 1H), 6.80-6.78 (d, 1H), 5.02 (s, 1H), 3.77 (s, 3H), 3.69 (s, 3H),3.62 (s, 3H); ¹³C-NMR (100 MHz) DMSO: 162.6, 159.0, 148.7, 146.0, 144.6,141.0, 129.8, 129.7, 125.9, 120.4, 119.0, 115.7, 115.4, 111.8, 109.8,94.7, 55.8, 55.7, 37.3, 34.4; LC/MS-MS: 407.1→341.1 m/z; GS1 and GS2 at30, DP=71, CE=33, CXP=22, t_(R)=3.9 min.6-amino-4-(4-fluorophenyl)-3-(4-methoxyphenyl)-1-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (22): A mixture of 4-fluorobenzaldehyde (300μL, 2.87 mmol), malononitrile (190 mg, 2.87 mmol) and TEA (400 μL, 2.87mmol, 1.0 equ.) in EtOH (10 mL) was stirred for 1.0 min, followed by theaddition of 8 (764 mg, 2.87 mmol). After 17 h, the precipitate formedwas filtered off and washed with EtOH and hexanes, and re-crystallizedfrom EtOH to afford 22 (800 mg, 1.83 mmol, 64% yield) as white solid.¹H-NMR (400 MHz) DMSO: 7.93-7.91 (d, 2H), 7.55-7.53 (m, 4H), 7.41-7.37(t, 1H), 7.26-7.23 (m, 4H), 7.07-7.05 (t, 2H), 6.84-6.82 (d, 2H), 5.11(s, 1H), 3.72 (s, 3H); ¹³C-NMR (100 MHz) DMSO: 162.6, 159.0, 146.6,145.5, 140.7, 140.6, 137.9, 130.0, 129.9 (d), 128.3, 127.1, 125.0,121.1, 120.2, 115.5 (d), 114.1, 97.3, 59.6, 55.5, 37.0; LC/MS-MS:439.2→373.0 m/z; GS1 and GS2 at 30, DP=61, CE=35, CXP=24, t_(R)=4.3 min.6-amino-3-(3,4-dimethoxyphenyl)-4-(4-fluorophenyl)-1-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (23): A mixture of 4-fluorobenzaldehyde(70.0 μL, 0.675 mmol), malononitrile (45.0 mg, 0.675 mmol) and TEA (90.0μL, 0.675 mmol) in EtOH (3.0 mL) was stirred for 1.0 min, followed bythe addition of the 7 (200 mg, 0.675 mmol). The reaction mixture wasconcentrated after 19 h and the crude material was purified by columnchromatography (25% EtOAc in hexanes ramped to 100% EtOAc). The yellowsolid was further purified by re-crystallization from EtOH to give 23(164 mg, 0.350 mmol, 12% yield) as a white solid. ¹H-NMR (400 MHz)CDCl₃: 7.80-7.78 (d, 2H), 7.52-7.48 (t, 2H), 7.38-7.35 (t, 1H),7.25-7.21 (m, 2H), 7.05-6.95 (m, 4H), 6.75-6.73 (d, 1H), 4.91 (s, 1H),4.84 (s, 2H), 3.84 (s, 3H), 3.71 (s, 3H); ¹³C-NMR (100 MHz) CDCl₃:163.2, 157.8, 149.2, 148.7, 147.5, 144.9, 138.6, 137.4, 129.3, 129.1(d), 127.1, 125.0, 121.5, 119.8, 119.0, 115.8 (d), 110.8, 109.9, 96.6,64.0, 55.8, 55.7, 37.5; LC/MS-MS: 469.3→403.1 m/z; GS1 and GS2 at 30,DP=6, CE=35, CXP=26, t_(R)=4.2 min.6-amino-4-(4-fluorophenyl)-3-(4-methoxyphenyl)-1-methyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (24): A mixture of 4-fluorobenzaldehyde (300μL, 2.87 mmol), malononitrile (190 mg, 2.87 mmol) and TEA (400 μL, 2.87mmol) in EtOH (10 mL) was stirred for 1.0 min, followed by the additionof 9 (586 mg, 2.87 mmol). The reaction mixture was concentrated after 19h and the precipitate formed was washed with EtOH and hexanes,re-crystallized from EtOH gave 24 (350 mg, 0.930 mmol, 32% yield) as awhite solid. ¹H-NMR (400 MHz) DMSO: 7.43-7.40 (d, 2H), 7.20-7.16 (m,2H), 7.10 (s, 2H), 7.06-7.02 (t, 2H), 6.78-6.76 (d, 2H), 4.99 (s, 1H),3.75 (s, 3H), 3.69 (s, 3H); ¹³C-NMR (100 MHz) DMSO: 162.5, 159.1, 145.8,144.6, 141.0, 141.0, 129.8 (d), 127.8, 125.7, 120.5, 115.5 (d), 113.9,94.9, 59.7, 55.4, 37.4, 34.4; LC/MS-MS: 377.1→311.1 m/z; GS1 and GS2 at30, DP=66, CE=33, CXP=20, t_(R)=4.0 min.6-amino-4-(3,5-difluorophenyl)-1,3-dimethyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(25): A mixture of the 3,5-difluorobenzaldehyde (0.408 g, 2.87 mmol),malononitrile (0.190 g, 2.87 mmol) and TEA (0.40 mL, 2.87 mmol) in EtOH(10 mL) was stirred for 5 min, followed by the addition of the 2 (0.321g, 2.87 mmol). The reaction mixture was concentrated after 23 h and thecrude material was recrystallized from EtOH and washed with EtOH andn-hexanes to give 25 (291 mg, 0.963 mmol, 34% yield) as a white solid.¹H-NMR (400 MHz) DMSO: 7.15 (br-s, 2H), 7.09-7.05 (t, 1H), 6.92-6.89 (m,2H), 4.66 (s, 1H), 3.57 (s, 3H), 1.68 (s, 3H); ¹³C-NMR (100 MHz) DMSO:163.9 (d, CF), 163.8 (d, CF), 160.2, 149.2 (t), 144.8, 142.9, 120.4,111.2 (m), 102.9 (t), 95.4, 57.5, 37.1, 33.9, 12.8. LC/MS-MS:303.9→236.9 m/z; GS1 and GS2 at 30, DP=11, CE=31, CXP=16, t_(R)=4.19min.6-amino-4-(3,5-difluorophenyl)-1-methyl-3-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(26): A mixture of 3,5-difluorobenzaldehyde (0.408 g, 2.87 mmol),malononitrile (0.190 g, 2.87 mmol) and TEA (0.40 mL, 2.87 mmol) in EtOH(10 mL) was stirred for 10 min, followed by the addition of 3 (0.500 g,2.87 mmol, 1 equ.). The reaction mixture was concentrated after 23 h andthe crude material was recrystallized from EtOH and to give 26 (282 mg,0.77 mmol, 27% yield) as a white solid. ¹H-NMR (400 MHz) DMSO: 7.48-7.47(d, 2H), 7.24-7.17 (m, 5H), 6.97-6.92 (m, 1H), 6.87-6.85 (d, 2H), 5.11(s, 1H), 3.74 (s, 3H); ¹³C-NMR (100 MHz) DMSO: 163.5 (d, CF), 163.5 (d,CF), 159.5, 146.1, 144.7, 133.0, 128.6, 128.1, 126.6, 126.5, 120.2,111.3 (d), 102.8, 95.5, 58.5, 37.6, 34.6. LC/MS-MS: 365.1→299.0 m/z; GS1and GS2 at 30, DP=86, CE=27, CXP=20, t_(R)=4.38 min.6-amino-4-(4-methoxyphenyl)-3-methyl-1-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(27): A mixture of anisaldehyde (350 μL, 2.87 mmol), malononitrile (190mg, 2.87 mmol) and TEA (400 μL, 2.87 mmol) in EtOH (10 mL) was stirredfor 1.0 min, followed by the addition of 1 (500 mg, 2.87 mmol). Thereaction mixture was concentrated after 24 h and the precipitate waswashed with EtOH and hexanes, and re-crystallized from EtOH to give 27(800 mg, 78% yield, 2.23 mmol) as a white solid. ¹H-NMR (400 MHz) DMSO:7.80-7.78 (d, 2H), 7.51-7.47 (t, 2H), 7.32-7.28 (t, 1H), 7.18-7.16 (m,4H), 6.91-6.89 (d, 2H), 4.62 (s, 1H), 3.74 (s, 3H), 1.79 (s, 3H);¹³C-NMR (100 MHz) DMSO: 159.7, 158.6, 145.7, 144.2, 138.0, 136.0, 129.7,129.2, 126.5, 120.5, 120.3, 114.3, 99.3, 59.0, 55.4, 36.4, 13.0;LC/MS-MS: 359.2→293.0 m/z; GS1 and GS2 at 30, DP=71, CE=29, CXP=20,t_(R)=4.14 min.6-amino-4-(4-methoxyphenyl)-1,3-dimethyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(28): A mixture of anisaldehyde (350 μL, 2.87 mmol), malononitrile (190mg, 2.87 mmol) and TEA (400 μL, 2.87 mmol) in EtOH (10 mL) was stirredfor 1.0 min, followed by the addition of 2 (321 mg, 2.87 mmol). Thereaction mixture was concentrated after 24 h and the crude material waspurified by column chromatography (25% EtOAc in hexanes ramped to 100%EtOAc). The yellow solid was washed with EtOH and hexanes, andre-crystallized from EtOH to give 28 (370 mg, 1.25 mmol, 44% yield) as awhite solid. ¹H-NMR (400 MHz) CDCl₃: 7.12-7.10 (d, 2H), 6.85-6.83 (d,2H), 4.61 (s, 2H), 4.55 (s, 1H), 3.79 (s, 3H), 3.69 (s, 3H), 1.80 (s,3H); ¹³C-NMR (100 MHz) CDCl₃: 158.8, 157.9, 144.5, 144.4, 134.5, 128.8,119.3, 114.0, 96.4, 64.2, 55.2, 36.7, 33.7, 12.7; LC/MS-MS: 297.0→231.2m/z; GS1 and GS2 at 30, DP=61, CE=27, CXP=16, t_(R)=3.71 min.6-amino-4-(4-methoxyphenyl)-1-methyl-3-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(29): A mixture of anisaldehyde (350 μL, 2.87 mmol), malononitrile (190mg, 2.87 mmol) and TEA (400 μL, 2.87 mmol) in EtOH (10 mL) was stirredfor 1.0 min, followed by the addition of 3 (500 mg, 2.87 mmol). Thereaction mixture was concentrated after 24 h and the precipitate waswashed with EtOH and hexanes, and the product re-crystallized from EtOHto give 29 (210 mg, 20% yield, 0.586 mmol) as a white solid. ¹H-NMR (400MHz) DMSO: 7.50-7.48 (d, 2H), 7.21-7.17 (m, 3H), 7.05-7.02 (m, 4H),6.76-6.74 (d, 2H), 4.91 (s, 1H), 3.76 (s, 3H), 3.64 (s, 3H); ¹³C-NMR(100 MHz) DMSO: 158.9, 158.3, 146.0, 144.6, 136.9, 133.2, 128.9, 128.5,127.8, 126.4, 120.6, 114.1, 95.9, 60.3, 55.3, 37.5, 34.5; LC/MS-MS:359.2→293.0 m/z; GS1 and GS2 at 30, DP=66, CE=29, CXP=20, t_(R)=3.98min.6-amino-4-(4-methoxyphenyl)-1-methyl-3-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(30): A mixture of anisaldehyde (350 μL, 2.87 mmol), malononitrile (190mg, 2.87 mmol) and TEA (400 μL, 2.87 mmol) in EtOH (10 mL) is stirredfor 1.0 min, followed by the addition of 4 (678 mg, 2.87 mmol). Thereaction mixture was concentrated after 24 h and the precipitate waswashed with EtOH and hexanes. The product was re-crystallized from EtOHto afford 30 (1.05 g, 87% yield, 2.50 mmol) as a white solid. ¹H-NMR(400 MHz) DMSO: 7.94-7.92 (d, 2H), 7.63-7.61 (d, 2H), 7.57-7.53 (t, 2H),7.40-7.36 (t, 1H), 7.29-7.23 (m, 3H) 7.15 (s, 2H), 7.13-7.11 (d, 2H),6.78-6.76 (d, 2H), 5.02 (s, 1H), 3.65 (s, 3H); ¹³C-NMR (100 MHz) DMSO:158.9, 158.4, 146.7, 145.6, 137.9, 136.5, 132.6, 129.8, 129.0, 128.7,128.5, 127.2, 127.0, 121.2, 120.3, 114.2, 98.3, 60.2, 55.3, 37.1;LC/MS-MS: 421.2→355.0 m/z; GS1 and GS2 at 30, DP=81, CE=35, CXP=24,t_(R)=4.32 min.6-amino-3,4-bis(4-methoxyphenyl)-1-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(31): A mixture of anisaldehyde (350 μL, 2.87 mmol, 1.0 equ.),malononitrile (190 mg, 2.87 mmol, 1.0 equ.) and TEA (400 μL, 2.87 mmol,1.0 equ.) in EtOH (10 mL) was stirred for 1.0 min, followed by theaddition 8 (764 mg, 2.87 mmol). The reaction mixture was concentratedafter 24 h and the precipitate was washed with EtOH and hexanes, andthen re-crystallized from EtOH to give 31 (1.06 g, 2.35 mmol, 82% yield)as a white solid. ¹H-NMR (400 MHz) DMSO: 7.93-7.91 (d, 2H), 7.56-7.52(m, 4H), 7.38-7.35 (t, 1H), 7.15-7.11 (m, 4H), 6.83-6.78 (m, 4H), 4.98(s, 1H), 3.70 (s, 3H), 3.66 (s, 3H); ¹³C-NMR (100 MHz) DMSO: 159.5,158.9, 158.4, 146.6, 145.5, 137.9, 136.6, 129.8, 129.0, 128.3, 127.0,125.2, 121.0, 120.4, 114.2, 114.1, 97.7, 60.3, 55.5, 55.3, 37.1;LC/MS-MS: 452.3→89.1 m/z; GS1 and GS2 at 30, DP=36, CE=39, CXP=4,t_(R)=3.47 min.6-amino-3,4-bis(4-methoxyphenyl)-1-methyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(32): A mixture of anisaldehyde (350 μL, 2.87 mmol), malononitrile (190mg, 2.87 mmol) and TEA (400 μL, 2.87 mmol) in ethanol (10 mL) wasstirred for 1.0 min, followed by the addition of 9 (689 mg, 2.87 mmol).The reaction mixture was concentrated after 24 h and the precipitate waswashed with EtOH and hexanes, and then re-crystallized from EtOH to give32 (690 mg, 1.78 mmol, 62% yield) as a white solid. ¹H-NMR (400 MHz)DMSO: 7.42-7.40 (d, 2H), 7.05-7.03 (d, 2H), 7.00 (s, 2H), 6.78-6.75 (dd,4H), 4.86 (s, 1H), 3.73 (s, 3H), 3.68 (s, 3H), 3.66 (s, 3H); ¹³C-NMR(100 MHz) DMSO: 159.0, 158.9, 158.3, 145.9, 144.5, 136.9, 128.9, 127.7,125.9, 120.6, 114.1, 113.9, 95.3, 60.3, 55.4, 55.3, 37.5, 34.3;LC/MS-MS: 389.2→323.0 m/z; GS1 and GS2 at 30, DP=66, CE=29, CXP=22,t_(R)=3.94 min.6-amino-4-(3-fluoro-4-methoxyphenyl)-1,3-dimethyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(33): A mixture of the 3-fluoro-4-methoxybenzaldehyde (0.442 g, 2.87mmol), malononitrile (0.190 g, 2.87 mmol) and TEA (0.40 mL, 2.87 mmol)in EtOH (10 mL) was stirred for 5 min, followed by the addition of 2(0.321 g, 2.87 mmol). The reaction mixture was concentrated after 18 hand the crude material was recrystallized from EtOH and the solid waswashed with EtOH and n-hexanes to give 31 (475 mg, 1.51 mmol, 53% yield)as a light orange solid. ¹H-NMR (400 MHz) DMSO: 7.10-7.04 (m, 3H),6.95-6.93 (m, 2H), 4.53 (s, 1H), 3.79 (s, 3H), 3.56 (s, 3H), 1.65 (s,3H); ¹³C-NMR (100 MHz) DMSO: 159.9, 153.0 (d, CF), 146.4 (d), 144.6,142.9, 137.6 (d), 124.1, 120.6, 115.4 (d), 114.0, 96.2, 58.5, 56.4,36.6, 33.9, 12.8. LC/MS-MS: 315.0→248.9 m/z; GS1 and GS2 at 30, DP=66,CE=27, CXP=16, t_(R)=4.05 min.6-amino-1,3-dimethyl-4-(2,4,6-trimethoxyphenyl)-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(34): A mixture of the 2,4,6-trimethoxybenzaldehyde (0.563 g, 2.87mmol), malononitrile (0.190 g, 2.87 mmol) and TEA (0.40 mL, 2.87 mmol)in EtOH (10 mL) was stirred for 10 min, followed by the addition of 2(0.321 g, 2.87 mmol). The reaction mixture was concentrated after 26 h.The crude material was purified by column chromatography on SiO₂ (2%MeOH in DCM). The yellow solid was recrystallized from EtOH and washedwith EtOH and n-hexanes to give 34 (60 mg, 0.168 mmol, 6% yield) as ayellow solid. ¹H-NMR (400 MHz) DMSO: 6.72 (s, 2H), 6.20 (brs, 2H), 4.97(s, 1H), 3.72 (s, 6H), 3.53 (s, 6H), 1.65 (s, 3H); ¹³C-NMR (100 MHz)DMSO: 161.3, 160.1, 145.4, 142.1, 121.3, 111.7, 96.5, 93.1, 91.2, 57.0,56.6, 55.5, 33.7, 26.1, 12.2. LC/MS-MS: 357.1→189.0 m/z; GS1 and GS2 at30, DP=56, CE=29, CXP=12, t_(R)=4.07 min.6-amino-4-(3-fluoro-4-methoxyphenyl)-1-methyl-3-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (35): A mixture of3-fluoro-4-methoxybenz-aldehyde (0.442 g, 2.87 mmol), malononitrile(0.190 g, 2.87 mmol) and TEA (0.40 mL, 2.87 mmol) in EtOH (10 mL) wasstirred for 10 min, followed by the addition of 3 (0.500 g, 2.87 mmol).The reaction mixture was concentrated after 18 h and the crude materialwas recrystallized from EtOH to give 35 (348 mg, 0.927 mmol, 33% yield)as a light white solid. ¹H-NMR (400 MHz) DMSO: 7.50-48 (d, 2H),7.22-7.15 (m, 3H), 7.07 (brs, 2H), 6.97-6.89 (m, 3H), 4.96 (s, 1H), 3.74(s, 3H), 3.71 (s, 3H); ¹³C-NMR (100 MHz) DMSO: 159.1, 152,8 (d, CF),146.1 (d), 146.0, 144.6, 137.9 (d), 133.2, 128.6, 127.9, 126.5, 124.0,120.5, 115.2 (d), 113.9, 95.4, 59.7, 56.3, 37.2, 34.5. LC/MS-MS:377.1→311.1 m/z; GS1 and GS2 at 30, DP=66, CE=31, CXP=20, t_(R)=4.27min.6-amino-1,3-dimethyl-4-(4-(trifluoromethyl)phenyl)-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(36): A mixture of the 4-(trifluoromethyl)benzaldehyde (0.500 g, 2.87mmol), malononitrile (0.190 g, 2.87 mmol) and TEA (0.40 mL, 2.87 mmol)in EtOH (10 mL) was stirred for 10 min, followed by the addition of 2(0.321 g, 2.87 mmol). The reaction mixture was concentrated after 22 hand purified by column chromatography on SiO₂ (2% MeOH in DCM) to give36 (445 mg, 1.33 mmol, 46% yield) as a yellow solid. ¹H-NMR (400 MHz)DMSO: 7.28 (s, 4H), 7.10 (brs, 2H), 4.64 (s, 1H), 3.57 (s, 3H), 1.64 (s,3H); ¹³C-NMR (100 MHz) DMSO: 160.1, 147.6, 144.7, 143.9, 142.9, 129.9,122.3, 121.4, 120.6, 96.2, 58.2, 36.8, 33.9, 12.8. LC/MS-MS: 337.2→59.1m/z; GS1 and GS2 at 30, DP=26, CE=31, CXP=10, t_(R)=5.10 min.6-amino-1-methyl-3-phenyl-4-(4-(trifluoromethyl)phenyl)-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (37): A mixture of4-(trifluoromethyl)benzaldehyde (0.300 g, 1.72 mmol), malononitrile(0.114 g, 1.72 mmol) and TEA (0.240 mL, 1.72 mmol) in EtOH (6 mL) wasstirred for 1.0 min, followed by the addition of 3 (300 mg, 1.72 mmol).The reaction mixture was concentrated after 22 h and purified by twicecolumn chromatography on SiO₂ (2% MeOH in DCM) and then EtOAc:hexanes(1:1) to give 37 (120 mg, 0.301 mmol, 18% yield) as a light yellowsolid. ¹H-NMR (400 MHz) DMSO: 7.58-7.56 (d, 2H), 7.49-7.47 (d, 2H),7.37-7.35 (d, 2H), 7.20-7.17 (m, 5H), 5.17 (s, 1H), 3.77 (s, 3H);¹³C-NMR (100 MHz) DMSO: 159.4, 149.3, 146.1, 144.7, 133.0, 128.8, 128.6,128.0, 126.4, 126.0, 125.7 (d), 123.3, 120.3, 94.9, 59.0, 37.9, 34.6.LC/MS-MS: 397.1→331.0 m/z; GS1 and GS2 at 30, DP=96, CE=33, CXP=22,t_(R)=4.44 min.6-amino-1,3-dimethyl-4-(4-(trifluoromethoxy)phenyl)-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (38): A mixture of4-(trifluoromethoxy)benzaldehyde (0.546 g, 2.87 mmol), malononitrile(0.190 g, 2.87 mmol) and TEA (0.40 mL, 2.87 mmol) in EtOH (10 mL) wasstirred for 10 min, followed by the addition of 2 (0.321 g, 2.87 mmol).The reaction mixture was concentrated after 22 h and purified by columnchromatography on SiO₂ (2% MeOH in DCM) to give 38 (359 mg, 1.03 mmol,36% yield) as an orange solid. ¹H-NMR (400 MHz) DMSO: 7.68-7.66 (d, 2H),7.40-7.38 (d, 2H), 7.14 (brs, 2H), 4.71 (s, 1H), 3.58 (s, 3H), 1.64 (s,3H); ¹³C-NMR (100 MHz) DMSO: 160.2, 149.2, 144.7, 142.9, 129.0, 127.2,125.9, 124.1, 120.5, 95.8, 57.8, 37.2, 33.9, 12.8. LC/MS-MS: 352.0→335.1m/z; GS1 and GS2 at 30, DP=26, CE=9, CXP=24, t_(R)=4.31 min.6-amino-1-methyl-3-phenyl-4-(4-(trifluoromethoxy)phenyl)-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (39): A mixture of4-(trifluoromethoxy)benzaldehyde (0.327 g, 1.72 mmol), malononitrile(0.114 g, 1.72 mmol) and TEA (0.240 mL, 1.72 mmol) in EtOH (6 mL) wasstirred for 10 min, followed by the addition of 3 (300 mg, 1.72 mmol).The reaction mixture was concentrated after 22 h and purified by columnchromatography on SiO₂ (2% MeOH in DCM) to give 39 (174 mg, 0.421 mmol,25% yield) as yellow solid. ¹H-NMR (400 MHz) DMSO: 7.47-7.45 (d, 2H),7.26-7.24 (d, 2H), 7.20-7.14 (m, 7H), 5.08 (s, 1H), 3.76 (s, 3H);¹³C-NMR (100 MHz) DMSO: 159.3, 147.4, 146.0, 144.7, 144.1, 133.1, 129.8,128.6, 128.0, 126.5, 121.7, 121.2, 120.4, 95.3, 59.3, 37.4, 34.5.LC/MS-MS: 413.1→346.9 m/z; GS1 and GS2 at 30, DP=86, CE=33, CXP=24,t_(R)=4.49 min.6-amino-4-(4-cyanophenyl)-1,3-dimethyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(40): A mixture of 4-formylbenzonitrile (0.376 g, 2.87 mmol),malononitrile (0.190 g, 2.87 mmol) and TEA (0.40 mL, 2.87 mmol) in EtOH(10 mL) was stirred for 10 min, followed by the addition of 2 (0.321 g,2.87 mmol). The reaction mixture was concentrated after 18 h and thecrude material was purified by column chromatography on SiO₂ (2% MeOH inDCM). The yellow solid was washed with EtOH and n-hexanes, andrecrystallized from EtOH to give 40 (484 mg, 1.66 mmol, 58% yield) as awhite off solid. ¹H-NMR (400 MHz) DMSO: 7.78-7.76 (d, 2H), 7.38-7.36 (d,2H), 7.17 (brs, 2H), 4.70 (s, 1H), 3.57 (s, 3H), 1.63 (s, 3H); ¹³C-NMR(100 MHz) DMSO: 160.2, 150.1, 144.7, 142.9, 133.0, 129.2, 120.4, 119.2,110.2, 95.6, 57.5, 37.4, 33.9, 12.8. LC/MS-MS: 292.0→226.2 m/z; GS1 andGS2 at 30, DP=51, CE=31, CXP=14, t_(R)=3.90 min.6-amino-4-(4-cyanophenyl)-1-methyl-3-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(41): A mixture of 4-formylbenzonitrile (0.376 g, 2.87 mmol),malononitrile (0.190 g, 2.87 mmol) and TEA (0.40 mL, 2.87 mmol) in EtOH(10 mL) was stirred for 10 min, followed by the addition of 3 (500 mg,2.87 mmol). The reaction mixture was concentrated after 18 h and thecrude material was purified by recrystallization from EtOH and n-hexanesto give 41 (160 mg, 0.453 mmol, 16% yield) as a white off solid. ¹H-NMR(400 MHz) DMSO: 7.68-7.66 (d, 2H), 7.48-7.46 (d, 2H), 7.35-7.33 (d, 2H),7.22-7.19 (m, 5H), 5.17 (s, 1H), 3.77 (s, 3H); ¹³C-NMR (100 MHz) DMSO:159.4, 150.2, 146.1, 144.7, 133.0, 132.8, 129.1, 128.7, 128.1, 126.5,120.2, 119.1, 110.0, 94.7, 58.6, 38.0, 34.6. LC/MS-MS: 354.2→288.1 m/z;GS1 and GS2 at 30, DP=66, CE=33, CXP=20, t_(R)=4.21 min.6-amino-4-(4-isopropylphenyl)-1,3-dimethyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(42): A mixture of 4-isopropylbenzaldehyde (0.425 g, 2.87 mmol),malononitrile (0.190 g, 2.87 mmol) and TEA (0.40 mL, 2.87 mmol) in EtOH(10 mL) was stirred for 10 min, followed by the addition of 2 (0.321 g,2.87 mmol). The reaction mixture was concentrated after 22 h andprecipitate was filtered off and washed with EtOH to give 42 (163 mg,0.528 mmol, 18% yield) as a light yellow solid. ¹H-NMR (400 MHz) DMSO:7.16-7.14 (d, 2H), 7.05-7.03 (d, 2H), 6.99 (s, 2H), 4.50 (s, 1H), 3.56(s, 3H), 2.85-2.79 (m, 1H), 1.64 (s, 3H), 1.17-1.15 (d, 6H); ¹³C-NMR(100 MHz) DMSO: 160.0, 147.2, 144.6, 142.9, 141.9, 127.9, 126.7, 120.8,96.7, 58.8, 37.1, 33.9, 33.4, 24.3, 12.9. LC/MS-MS: 309.1→243.0 m/z; GS1and GS2 at 30, DP=71, CE=31, CXP=16, t_(R)=4.44 min.6-amino-4-(4-isopropylphenyl)-1-methyl-3-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(43): A mixture of 4-isopropylbenzaldehyde (0.254 g, 1.72 mmol),malononitrile (0.114 g, 1.72 mmol) and TEA (0.240 mL, 1.72 mmol) inethanol (6 mL) was stirred for 10 min, followed by the addition of 3(0.300 g, 1.72 mmol). The reaction mixture was concentrated after 22 hand purified by column chromatography on SiO₂ twice (2% MeOH in DCM) andthen EtOAc:Hexanes; 1:1) to give 43 (207 mg, 0.559 mmol, 33% yield) as ayellow solid. ¹H-NMR (400 MHz) DMSO: 7.50-7.48 (d, 2H), 7.22-7.16 (m,3H), 7.09-7.02 (m, 6H), 4.92 (s, 1H), 3.76 (s, 3H), 2.80-2.73 (m, 1H),1.12-1.10 (d, 6H); ¹³C-NMR (100 MHz) DMSO: 159.2, 147.1, 146.1, 144.5,142.3, 133.3, 128.6, 127.9, 127.7, 126.7, 126.4, 120.7, 95.9, 60.1,37.9, 34.5, 33.3, 24.2. LC/MS-MS: 371.1→305.0 m/z; GS1 and GS2 at 30,DP=106, CE=29, CXP=20, t_(R)=4.60 min.6-amino-4-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3-dimethyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (44): A mixture of the1,4-benzodioxan-6-carboxaldehyde (0.471 g, 2.87 mmol), malononitrile(0.190 g, 2.87 mmol) and TEA (0.40 mL, 2.87 mmol) in EtOH (10 mL) wasstirred for 5 min, followed by the addition of 2 (0.321 g, 2.87 mmol).The reaction mixture was concentrated after 23 h and the crude materialwas recrystallized from EtOH and washed with EtOH and n-hexanes to give44 (131 mg, 0.404 mmol, 14% yield) as a yellow solid. ¹H-NMR (400 MHz)DMSO: 6.98 (brs, 2H), 6.76-6.74 (d, 1H), 6.59-6.57 (m, 2H), 4.43 (s,1H), 4.18 (s, 4H), 3.56 (s, 3H), 1.67 (s, 3H); ¹³C-NMR (100 MHz) DMSO:159.9, 144.6, 143.5, 143.0, 142.6, 137.7, 120.7, 120.7, 117.3, 116.5,96.6, 64.5, 64.4, 58.9, 36.8, 33.9, 12.9. LC/MS-MS: 325.0→259.1 m/z; GS1and GS2 at 30, DP=51, CE=29, CXP=18, t_(R)=3.98 min.6-amino-4-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-methyl-3-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(45): A mixture of 1,4-benzodioxan-6-carboxaldehyde (0.471 g, 2.87mmol), malononitrile (0.190 g, 2.87 mmol) and TEA (0.40 mL, 2.87 mmol)in EtOH (10 mL) was stirred for 10 min, followed by the addition of 3(0.500 g, 2.87 mmol). The reaction mixture was concentrated after 23 hand the crude material was purified by column chromatography on SiO₂ (2%MeOH in DCM) and then recrystallized from EtOH to give 45 (93 mg, 0.240mmol, 8% yield) as a yellow solid. ¹H-NMR (400 MHz) DMSO: 7.53-7.51 (d,2H), 7.25-7.18 (m, 3H), 7.03 (brs, 2H), 6.98-6.66 (d, 1H), 6.60-6.57 (m,2H), 4.86 (s, 1H), 4.13 (s, 4H), 3.75 (s, 3H); ¹³C-NMR (100 MHz) DMSO:159.1, 146.0, 144.5, 143.5, 142.5, 138.1, 133.3, 128.7, 127.9, 126.5,120.6, 120.5, 117.2, 116.3, 95.8, 64.4, 64.3, 60.2, 37.5, 34.5.LC/MS-MS: 387.1→321.0 m/z; GS1 and GS2 at 30, DP=66, CE=31, CXP=22,t_(R)=4.25 min.6-amino-1,3-dimethyl-4-(pyridin-4-yl)-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(46): A mixture of 3-pyridincarboxaldehyde (0.307 g, 2.87 mmol),malononitrile (0.190 g, 2.87 mmol) and TEA (0.40 mL, 2.87 mmol) inethanol (10 mL) was stirred for 10 min, followed by the addition of 2(0.321 g, 2.87 mmol). The reaction mixture was concentrated after 22 hand purified by column chromatography on SiO₂ (2% MeOH in DCM) andwashed with EtOH to give 46 (132 mg, 0.493 mmol, 17% yield) as a lightorange solid. ¹H-NMR (400 MHz) DMSO: 8.49-8.48 (d, 2H), 7.18-7.18 (m,4H), 4.61 (s, 1H), 3.58 (s, 3H), 1.66 (s, 3H); ¹³C-NMR (100 MHz) DMSO:160.4, 152.9, 150.3, 144.8, 142.9, 123.4, 120.4, 95.2, 57.1, 36.8, 33.9,12.8. LC/MS-MS: 268.0→189.1 m/z; GS1 and GS2 at 30, DP=56, CE=25,CXP=12, t_(R)=3.38 min.6-amino-1-methyl-3-phenyl-4-(pyridin-4-yl)-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(47): A mixture of 3-pyridincarboxaldehyde (0.307 g, 2.87 mmol),malononitrile (0.190 g, 2.87 mmol) and TEA (0.40 mL, 2.87 mmol) in EtOH(10 mL) was stirred for 10 min, followed by the addition of 3 (500 mg,2.87 mmol). After 22 h the precipitate was filtered off and washed withEtOH and hexanes to give 47 (340 mg, 1.03 mmol, 36% yield) as a whitesolid. ¹H-NMR (400 MHz) DMSO: 8.38-8.36 (dd, 2H), 7.48-7.46 (d, 2H),7.22-7.13 (m, 7H), 5.08 (s, 1H), 3.77 (s, 3H); ¹³C-NMR (100 MHz) DMSO:159.6, 153.0, 150.1, 146.1, 144.7, 133.0, 128.7, 128.1, 126.5, 123.2,120.2, 94.3, 58.2, 37.4, 34.6. LC/MS-MS: 330.1→80.1 m/z; GS1 and GS2 at30, DP=76, CE=63, CXP=4, t_(R)=3.94 min.6-amino-1,3-dimethyl-4-(pyridin-3-yl)-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(48): A mixture of 3-pyridincarboxaldehyde (0.307 g, 2.87 mmol),malononitrile (0.190 g, 2.87 mmol) and TEA (0.40 mL, 2.87 mmol) inethanol (10 mL) was stirred for 5 min, followed by the addition of 2(0.321 g, 2.87 mmol). The reaction mixture was concentrated after 22 hand the precipitate was filtered off and washed with EtOH to give 48(463 mg, 1.73 mmol, 60% yield) as a white solid. ¹H-NMR (400 MHz) DMSO:8.43-8.43 (d, 2H), 7.53-7.51 (d, 1H), 7.34-7.31 (m, 1H), 7.14 (brs, 2H),4.63 (s, 1H), 3.57 (s, 3H), 1.64 (s, 3H); ¹³C-NMR (100 MHz) DMSO: 160.2,149.4, 148.8, 144.8, 142.8, 139.7, 135.8, 124.2, 120.5, 95.7, 57.8,35.0, 33.9, 12.8. LC/MS-MS: 268.0→189.2 m/z; GS1 and GS2 at 30, DP=71,CE=34, CXP=12, t_(R)=3.5 min.6-amino-1-methyl-3-phenyl-4-(pyridin-3-yl)-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(49): A mixture of 3-pyridincarboxaldehyde (0.307 g, 2.87 mmol),malononitrile (0.190 g, 2.87 mmol) and TEA (0.40 mL, 2.87 mmol) in EtOH(10 mL) was stirred for 10 min, followed by the addition of 3 (500 mg g,2.87 mmol). A white precipitate was formed and filtered off after 22 h.The formed precipitate was recrystallized with EtOH to give 49 (389 mg,1.19 mmol, 41% yield) as a white solid. ¹H-NMR (400 MHz) DMSO: 8.41-8.40(d, 1H), 8.30-8.29 (dd, 1H), 7.49-7.47 (d, 3H), 7.23-7.16 (m, 6H), 5.12(s, 1H), 3.77 (s, 3H); ¹³C-NMR (100 MHz) DMSO: 159.4, 149.3, 148.5,146.1, 144.7, 139.9, 135.6, 133.0, 128.6, 128.0, 126.5, 124.0, 120.4,94.8, 58.9, 35.6, 34.6. LC/MS-MS: 330.1→80.1 m/z; GS1 and GS2 at 30,DP=56, CE=57, CXP=14, t_(R)=3.96 min.6-amino-4-(6-bromopyridin-2-yl)-1,3-dimethyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(50): A mixture of 6-bromo-2-pyridincarboxaldehyde (0.307 g, 2.87 mmol),malononitrile (0.190 g, 2.87 mmol) and TEA (0.40 mL, 2.87 mmol) in EtOH(10 mL) was stirred for 10 min, followed by the addition of 2 (0.321 g,2.87 mmol). The reaction mixture was filtered after 22 h and theprecipitate was filtered off and washed with EtOH to give 50 (427 mg,1.23 mmol, 43% yield) as a white solid. ¹H-NMR (400 MHz) DMSO: 7.73-7.69(t, 1H), 7.50-7.48 (d, 1H), 7.32-7.30 (d, 1H), 7.19 (brs, 2H), 4.69 (s,1H), 3.56 (s, 3H), 1.71 (s, 3H); ¹³C-NMR (100 MHz) DMSO: 164.4, 160.7,144.7, 142.9, 141.4, 140.8, 127.0, 122.0, 120.5, 95.3, 56.3, 39.3, 33.9,12.8. LC/MS-MS: 348.0→283.0 m/z; GS1 and GS2 at 30, DP=66, CE=25,CXP=18, t_(R)=4.00 min.6-amino-4-(6-bromopyridin-2-yl)-1-methyl-3-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(51): A mixture of 6-bromo-2-pyridincarboxaldehyde (0.307 g, 2.87 mmol),malononitrile (0.190 g, 2.87 mmol) and TEA (0.40 mL, 2.87 mmol) in EtOH(10 mL) was stirred for 10 min, followed by the addition of 3 (500 g,2.87 mmol). The reaction mixture was filtered after 22 h and theprecipitate was filtered off and washed with EtOH to give 51 (971 mg,2.38 mmol, 83% yield) as a white solid. ¹H-NMR (400 MHz) DMSO: 7.59-7.53(t, 1H), 7.52-7.50 (d, 2H), 7.38-7.35 (d, 1H), 7.30-7.26 (d, 1 H),7.24-7.21 (m, 5H), 5.12 (s, 1H), 3.76 (s, 3H); ¹³C-NMR (100 MHz) DMSO:164.4, 160.0, 146.0, 144.7, 141.2, 140.5, 133.0, 128.6, 128.1, 126.9,126.4, 122.2, 120.2, 94.6, 57.5, 39.3, 34.5. LC/MS-MS: 408.0→175.1 m/z;GS1 and GS2 at 30, DP=86, CE=31, CXP=10, t_(R)=4.28 min.4-(4-(1H-imidazol-1-yl)phenyl)-6-amino-1,3-dimethyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (52): A mixture of the4-(1H-imidazol-1-yl)benzaldehyde (0.494 g, 2.87 mmol), malononitrile(0.190 g, 2.87 mmol) and TEA (0.40 mL, 2.87 mmol) in EtOH (10 mL) wasstirred for 1.0 min, followed by the addition of the 2 (0.321 g, 2.87mmol). The reaction mixture was concentrated after 22 h and purified bycolumn chromatography on SiO₂ (2% MeOH in DCM) to give 52 (322 mg, 0.967mmol, 34% yield) as a light brown solid. ¹H-NMR (400 MHz) DMSO: 8.20 (s,1H), 7.69 (s, 1H), 7.58-7.56 (d, 2H), 7.29-7.27 (d, 2H), 7.09-7.07 (d,3H), 4.64 (s, 1H), 3.58 (s, 3H), 1.68 (s, 3H); ¹³C-NMR (100 MHz) DMSO:160.1, 144.7, 143.2, 143.0, 136.1, 136.0, 130.3, 129.5, 121.0, 120.7,118.5, 95.3, 58.4, 36.9, 33.9, 12.9. LC/MS-MS: 333.3→266.9 m/z; GS1 andGS2 at 30, DP=61, CE=41, CXP=18, t_(R)=3.4 min.4-(4-(1H-imidazol-1-yl)phenyl)-6-amino-1-methyl-3-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (53): A mixture4-(1H-imidazol-1-yl)benzaldehyde (0.296 g, 1.72 mmol), malononitrile(0.114 g, 1.72 mmol) and TEA (0.240 mL, 1.72 mmol) in EtOH (6 mL) wasstirred for 10 min, followed by the addition of 3 (0.300 g, 1.72 mmol).The reaction mixture was concentrated after 22 h and purified by columnchromatography on SiO₂ (2% MeOH in DCM) to give 53 (308 mg, 0.780 mmol,45% yield) as a white off solid. ¹H-NMR (400 MHz) DMSO: 8.15 (s, 1H),7.64 (s, 1H), 7.54-7.52 (d, 2H), 7.49-7.47 (d, 2H), 7.28-7.13 (m, 7H),7.04 (s, 1H), 5.09 (s, 1H), 3.78 (s, 3H); ¹³C-NMR (100 MHz) DMSO: 159.2145.1, 144.6, 143.5, 135.9, 135.9, 133.2, 130.2, 129.3, 128.7, 128.0,126.5, 120.7, 120.5, 118.4, 95.4, 59.6, 37.6, 34.6. LC/MS-MS:395.2→144.0 m/z; GS1 and GS2 at 30, DP=101, CE=59, CXP=8, t_(R)=3.89min.

6-amino-4-(5-bromothiophen-2-yl)-1,3-dimethyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(54): A mixture of the 5-bromothiophene-2-carbaldehyde (0.548 g, 2.87mmol), malononitrile (0.190 g, 2.87 mmol) and triethylamine (0.40 mL,2.87 mmol) in ethanol (10 mL) was stirred for 10 min, followed by theaddition of 2 (0.321 g, 2.87 mmol). The reaction mixture was filteredafter 22 h and precipitate was recrystallized with ethanol to give 54(221 mg, 0.628 mmol, 22% yield) as a light orange solid. ¹H-NMR (400MHz) DMSO: 7.18 (s, 2H), 7.02-7.02 (d, 1H), 6.86-6.85 (d, 1H), 4.93 (s,1H), 3.56 (s, 3H), 1.81 (s, 3H); ¹³C-NMR (100 MHz) DMSO: 159.9, 151.9,144.2, 143.1, 130.3, 126.0, 120.3, 110.8, 95.8, 58.3, 33.9, 33.2, 12.8.LC/MS-MS: 352.9→287.0 m/z; GS1 and GS2 at 30, DP=66, CE=29, CXP=18,t_(R)=4.3 min.

6-amino-4-(5-bromothiophen-2-yl)-1-methyl-3-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (55): A mixture of5-bromothiophene-2-carbaldehyde (0.328 g, 1.72 mmol), malononitrile(0.114 g, 1.72 mmol) and triethylamine (0.240 mL, 1.72 mmol) in ethanol(6 mL) was stirred for 10 min, followed by the addition of 3 (0.300 g,1.72 mmol). The reaction mixture was concentrated after 22 h andpurified by column chromatography on SiO₂ (2% MeOH in DCM) to give 55(80 mg, 0.194 mmol, 11% yield) as orange solid. ¹H-NMR (400 MHz) DMSO:7.61-7.59 (d, 2H), 7.30-7.23 (m, 5H), 6.92-6.91 (d, 1H), 6.78-6.77 (d,1H), 5.41 (s, 1H), 3.74 (s, 3H); ¹³C-NMR (100 MHz) DMSO: 159.3, 151.9,145.4, 144.8, 133.0, 130.3, 128.8, 128.2, 126.6, 125.8, 120.2, 110.4,95.2, 59.1, 34.5, 33.9. LC/MS-MS: 415.1→348.0 m/z; GS1 and GS2 at 30,DP=81, CE=31, CXP=22, t_(R)=4.5 min.

The foregoing examples of the present invention have been presented forpurposes of illustration and description. Furthermore, these examplesare not intended to limit the invention to the form disclosed herein.Consequently, variations and modifications commensurate with theteachings of the description of the invention, and the skill orknowledge of the relevant art, are within the scope of the presentinvention. The specific embodiments described in the examples providedherein are intended to further explain the best mode known forpracticing the invention and to enable others skilled in the art toutilize the invention in such, or other, embodiments and with variousmodifications required by the particular applications or uses of thepresent invention. It is intended that the appended claims be construedto include alternative embodiments to the extent permitted by the priorart.

What is claimed is:
 1. A compound having a chemical structure:

R₁-R₇ are independently selected from hydrogen, halogen, —OH, C₁-C₁₂alkyl, C₃-C₁₂ alkenyl, C₄-C₁₂ dienyl, C₆-C₁₂ trienyl, C₈-C₁₂ tetraenyl,C₆-C₁₂ aryl, substituted C₆-C₁₂ aryl, C₁-C₁₂-alkoxy, carboxy, cyano,C₁-C₁₂ alkanoyloxy, C₁-C₁₂ alkylthio, C₁-C₁₂ alkylsulfonyl, C₂-C₁₂alkoxycarbonyl, C₂-C₁₂ alkanoylamino, —O—R₈,—S—R₈, —SO₂—R₈, —NHSO₂R₈,and —NHCO₂R₈; and R₈ is a C₁-C₁₂ alkyl substituted with one to threegroups selected from halogen, oxygen, C₁-C₆ alkyl, C₆-C₁₀ aryl, andC₁-C₆ alkoxy, wherein at least one of R₃-R₇ is: a C₁-C₁₂ alkylsubstituted with halogen, —O—R₈, —S—R₈, —SO₂—R₈, —NHSO₂R₈, and —NHCO₂R₈,wherein R₈ is a C₁-C₁₂ alkyl substituted with halogen, or R₃ and R₄together form cyclohexane, 1,4-dioxane, or phenyl, and pharmaceuticallyacceptable enantiomers, diastereomers, racemates, and salts thereof. 2.The compound of claim 1, wherein R₃ is alkyl or alkoxy substituted withhalogen.
 3. The compound of claim 1, wherein R₃ is —CF₃ or —OCF₃.
 4. Thecompound of claim 1, wherein R₄ is alkyl or alkoxy substituted withhalogen.
 5. The compound of claim 1, wherein R₄ is —CF₃ or —OCF₃.
 6. Thecompound of claim 1, wherein R₅ is alkyl or alkoxy substituted withhalogen.
 7. The compound of claim 1, wherein R₅ is —CF₃ or —OCF₃.
 8. Thecompound of claim 1, wherein R₆ is alkyl or alkoxy substituted withhalogen.
 9. The compound of claim 1, wherein R₆ is —CF₃ or —OCF₃. 10.The compound of claim 1, wherein R₇ is alkyl or alkoxy substituted withhalogen.
 11. The compound of claim 1, wherein R₇ is —CF₃ or —OCF₃. 12.The compound of claim 1, wherein R₃ and R₄ together form cyclohexane,1,4-dioxane, or phenyl.
 13. A pharmaceutical composition comprising acompound of claim 1 and a pharmaceutically acceptable carrier.
 14. Thepharmaceutical composition of claim 13, wherein the pharmaceuticalcomposition is mono-phasic and suitable for parenteral or oraladministration of a therapeutically-effective amount of the compound.15. A method of treating or ameliorating cancer, or preventingmetastasis of a cancer in a subject suffering from cancer, comprising:administering a therapeutically-effective amount of the compound ofclaim
 1. 16. The method of claim 15, wherein the compound isadministered to the subject within a pharmaceutical composition.
 17. Themethod of claim 16, wherein the pharmaceutical composition ismono-phasic and suitable for parenteral or oral administration of atherapeutically-effective amount of the compound.
 18. The method ofclaim 15, wherein the pharmaceutical composition is administered inconjunction with one or more geranylgeranyltransferase type I (GGTase-I)inhibitors, surgery, chemotherapy, radiation, immunotherapy, orcombinations thereof.